Spider silk fusion protein structures for binding to an organic target

ABSTRACT

A protein structure capable of selective interaction with an organic target is provided. The protein structure is a polymer comprising as a repeating structural unit a recombinant fusion protein that is capable of selective interaction with the organic target. The fusion protein is comprising the moieties B, REP and CT, and optionally NT. B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target. REP is a moiety of from 70 to 300 amino acid residues and is derived from the repetitive fragment of a spider silk protein. CT is a moiety of from 70 to 120 amino acid residues and is derived from the C-terminal fragment of a spider silk protein. NT is an optional moiety of from 100 to 160 amino acid residues and is derived from the N-terminal fragment of a spider silk protein. The fusion protein and protein structure thereof is useful as an affinity medium and a cell scaffold material.

This application is a Continuation of U.S. patent application Ser. No.13/880,628 filed on Aug. 6, 2013, which is the National Phase of PCTInternational Application No. PCT/EP2011/068626 filed on Oct. 25, 2011and claims priority under 35 U.S.C. §119(a) to patent application Ser.No. 10/189,059.8 filed in Europe on Oct. 27, 2010, all of which arehereby expressly incorporated by reference into the present application.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of recombinant fusionproteins, and more specifically to fusion proteins comprising moietiesderived from spider silk proteins (spidroins). The present inventionprovides methods for providing a protein structure which is a polymercomprising a recombinant fusion protein, which is comprising moietiesderived from spidroins. There is also provided novel protein structuresfor binding to an organic target.

BACKGROUND TO THE INVENTION

In applied protein chemistry, it is a common problem how to formulate orpresent a biologically active peptide or protein to the relevant site ofactivity, typically an organic target, such as a nucleic acid, aprotein, a complex of proteins, or a complex of a protein(s) and/orlipids and/or carbohydrates and/or a nucleic acid(s). The simplestsolution is simply to provide an aqueous solution of the biologicallyactive peptide or protein. Many applications do however require somefurther means to achieve the desired goal. For instance, thepeptides/proteins may be associated with a lipid mixture or chemicallyimmobilized to a support structure.

Applications for peptides/proteins immobilized to a support structureinclude preparative and analytical separation procedures, such asbioprocesses, chromatography, cell capture and culture, active filters,and diagnostics. Structures based on extracellular matrix proteins, e.g.collagen, are disclosed in EP 704 532 and EP 985 732.

It has also been suggested to use spider silk proteins in a supportingstructure. Spider silks are nature's high-performance polymers,obtaining extraordinary toughness and extensibility due to a combinationof strength and elasticity. Spiders have up to seven different glandswhich produce a variety of silk types with different mechanicalproperties and functions. Dragline silk, produced by the major ampullategland, is the toughest fiber. It consists of two main polypeptides,mostly referred to as major ampullate spidroin (MaSp) 1 and 2, but e.g.as ADF-3 and ADF-4 in Araneus diadematus. These proteins have molecularmasses in the range of 200-720 kDa. Spider dragline silk proteins, orMaSps, have a tripartite composition; a non-repetitive N-terminaldomain, a central repetitive region comprised of many iteratedpoly-Ala/Gly segments, and a non-repetitive C-terminal domain. It isgenerally believed that the repetitive region forms intermolecularcontacts in the silk fibers, while the precise functions of the terminaldomains are less clear. It is also believed that in association withfiber formation, the repetitive region undergoes a structural conversionfrom random coil and α-helical conformation to β-sheet structure. TheC-terminal region of spidroins is generally conserved between spiderspecies and silk types.

WO 07/078239 and Stark, M. et al., Biomacromolecules 8: 1695-1701,(2007) disclose a miniature spider silk protein consisting of arepetitive fragment with a high content of Ala and Gly and a C-terminalfragment of a protein, as well as soluble fusion proteins comprising thespider silk protein. Fibers of the spider silk protein are obtainedspontaneously upon liberation of the spider silk protein from its fusionpartner.

Rising, A. et al., CMLS 68(2): 169-184 (2010) reviews advances in theproduction of spider silk proteins.

US 2009/0263430 discloses chemical coupling of the enzymeβ-galactosidase to films of a miniature spider silk protein. However,chemical coupling may require conditions which are unfavourable forprotein stability and/or function. Proteins containing multiple repeatsof a segment derived from the repetitive region of spider silk proteinshave been designed to include a RGD cell-binding segment (Bini, E etal., Biomacromolecules 7:3139-3145 (2006)) and/or a R5 peptide (Wong PoFoo, C et al., Proc Natl Acad Sci 103 (25): 9428-9433 (2006)) or otherprotein segments involved in mineralization (Huang, J et al.,Biomaterials 28: 2358-2367 (2007); WO 2006/076711). In these prior artdocuments, films are formed by solubilizing the fusion proteins in thedenaturing organic solvent hexafluoroisopropanol (HFIP) and drying.

US 2005/261479 A1 discloses a method of for purification of recombinantsilk proteins having an affinity tag, involving magnetic affinityseparation of individual silk proteins from complex mixtures withoutformation of silk protein fibers or other polymer structures.

Known supporting structures and associated techniques have certaindrawbacks with regard to e.g. economy, efficiency, stability,regenerating capacity, bioactivity and biocompatibility.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel proteinstructure that is capable of selective interaction with an organictarget.

It is also an object of the present invention to provide a proteinstructure that is capable of selective interaction with an organictarget, wherein the structure is formed without use of harsh solventswhich may have an unpredictable effect on the secondary structure oractivity of the protein and/or remain in the protein structure.

It is one object of the present invention to provide a stable proteinstructure that is capable of selective interaction with an organictarget, which protein structure can readily be regenerated after use,e.g. with chemical treatment.

It is another object of the present invention to provide a stableprotein structure that is biocompatible and suitable for cell cultureand as an implant.

It is yet another object of the invention to provide a protein structurewith a high density of evenly spaced functionalities that are capable ofselective interaction with an organic target.

It is a further object of the invention to provide a protein structurewhich maintains its selective binding ability upon storage at +4° C. orat room temperature for months.

It is also an object of the invention to provide a protein structurewhich is autoclavable, i.e. maintains its selective binding abilityafter sterilizing heat treatment.

For these and other objects that will be evident from the followingdisclosure, the present invention provides according to a first aspect afusion protein and a protein structure consisting of polymers comprisingas a repeating structural unit the fusion protein as set out in theclaims.

According to a related aspect, the present invention provides anisolated polynucleic acid encoding the fusion protein and a method ofproducing the fusion protein as set out in the claims.

The present invention provides according to another aspect a method forproviding a protein structure as set out in the claims.

The present invention provides according to a further aspect an affinitymedium as set out in the claims.

The present invention provides according to one aspect a cell scaffoldmaterial as set out in the claims. According to a related aspect, thepresent invention also provides a combination of cells and a cellscaffold material according to the claims.

The present invention provides according to an aspect novel uses of aprotein structure and a fusion protein as set out in the claims.

The present invention provides according to another aspect a method forseparation of an organic target from a sample as set out in the claims.

The present invention provides according to a further aspect a methodfor immobilization and optionally cultivation of cells as set out in theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of spidroin C-terminal domains.

FIG. 2 shows a sequence alignment of spidroin N-terminal domains.

FIG. 3 shows a macroscopic fiber of a fusion protein comprising a Zdomain.

FIG. 4 shows a SDS-PAGE gel from purification and analysis of a fusionprotein comprising a Z domain.

FIG. 5 shows fibers made of fusion proteins and control fibers thatillustrate functionality of the Z domain in a fusion protein.

FIG. 6 shows part of a casted film made of fusion proteins at the bottomof a tissue culture plate.

FIGS. 7-12 show non-reducing SDS-PAGE gels illustrating thefunctionality of the Z domain in fusion protein structures.

FIGS. 13-15 show non-reducing SDS-PAGE gels illustrating the IgG-bindingcapacity of the Z domain in a fusion protein structures compared to acommercial protein A matrix.

FIGS. 16-17 show non-reducing SDS-PAGE gels of cleaning-in-place (CIP)procedures of fusion protein structures compared to a commercial proteinA matrix.

FIG. 18 shows fluorescence intensities from protein films soaked withbiotinylated Atto-565.

FIG. 19 shows a graph and a linear fit of fluorescence intensity valuesfor different concentrations of biotinylated Atto-565 upon binding to afusion protein film.

FIG. 20 shows graphs of fluorescence intensity values before (−) andafter (+) addition of biotinylated Atto-565 to wells with films made offusion protein or control.

FIG. 21 is a graph showing a standard curve and a linear fit ofresulting reaction velocities in catalysis by biotinylated HRP free insolution.

FIG. 22 is a graph showing the reaction velocity in catalysis bybiotinylated HRP immobilised to a fusion protein film compared tocontrol.

FIG. 23 shows a reducing SDS-PAGE gel of solubilized fusion proteinstructures.

FIG. 24-26 shows graphs illustrating binding of IgG-HRP to a fusionprotein film comprising Z domains.

FIG. 27 shows graphs illustrating binding of IgG-Alexa Fluor 633 to afusion protein film comprising Z domains.

FIG. 28 shows a non-reducing SDS-PAGE gel illustrating the functionalityof the Z domain in a fusion protein after autoclaving.

FIG. 29 shows a SDS-PAGE gel of cleavage products from Protease 3Ctreatment of a fusion protein fiber comprising Z domains.

FIG. 30 shows a non-reducing SDS-PAGE gel illustrating the functionalityof the Z domain in fusion protein structures formed in the presence ofan organic target (IgG).

FIG. 31-32 shows non-reducing SDS-PAGE gels illustrating thefunctionality of the Abd domain in fusion protein structures.

FIG. 33-34 shows non-reducing SDS-PAGE gels illustrating thefunctionality of the C2 domain in fusion protein structures.

List of appended sequences SEQ ID NO 1 4Rep 2 4RepCT 3 NT4Rep 4 NT5Rep 5NT4RepCTHis 6 NT 7 CT 8 consensus NT sequence 9 consensus CT sequence 10repetitive sequence from Euprosthenops australis MaSp1 11 consensus Gsegment sequence 1 12 consensus G segment sequence 2 13 consensus Gsegment sequence 3 14 HisZQG4Rep4CT 15 HisZQG4Rep4CT (DNA) 16HisAbdQG4RepCT 17 HisAbdQG4RepCT (DNA) 18 HisC2QG4RepCT 19 HisC2QG4RepCT(DNA) SEQ ID NO 20 4RepCT 2 21 4RepCT 2 (DNA) 22 M44RepCT 23 M44RepCT(DNA) 24 modM44RepCT 25 modM44RepCT (DNA) 26 4RepCTM4 27 4RepCTM4 (DNA)

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally based on the insight that solidprotein structures capable of selective interaction with an organictarget can be prepared in the form of polymers of a recombinant fusionprotein as a repeating structural unit. The fusion protein is comprisingat least one non-spidroin moiety of more than 30 amino acid residuesthat is capable of selective interaction with the organic target, andmoieties corresponding to at least the repetitive and the C-terminalfragments of a spider silk protein. Surprisingly, the moieties derivedfrom the spider silk protein can be induced to rearrange structurallyand as a result form polymeric, solid structures, while the non-spidroinmoiety is not structurally rearranged but maintains its desirablestructure and function, i.e. capability of selective interaction withthe organic target. The protein structures can be obtained without achemical coupling step or a denaturing method step, which facilitatesthe procedure and improves the chances of obtaining a fusion proteinwith maintained functionality of its moieties, in particular when thefunctions are dependent on the secondary structure of the moieties. Theformation of these fusion protein polymers can be tightly controlled,and this insight has been developed into further novel proteinstructures, methods of producing the protein structures and uses of theprotein structures in various applications and methods.

The fusion protein according to the invention thus harbors both thedesired selective interaction activity and an internal solid supportactivity that is employed in the protein structure under physiologicalconditions. It must be considered as surprising that the bindingactivity of the fusion protein is maintained although the non-spidroinmoiety is covalently attached to the spidroin moiety when the latter isstructurally rearranged to form polymeric, solid structures. In fact,the heat and/or chemical stability and/or binding activity of the moietyproviding the selective interaction activity may be increased whenintegrated in a fusion protein structure according to the invention. Theprotein structure also provides a high and predictable density of theselective interaction activity towards an organic target. Losses ofvaluable protein moieties with selective interaction activity areminimized, since all expressed protein moieties are associated with thesolid support.

The polymers which are formed from the fusion proteins according to theinvention are solid structures and are useful for their physicalproperties, especially the useful combination of high strength,elasticity and light weight. A particularly useful feature is that thespidroin-derived moieties of the fusion protein are biochemically robustand suitable for regeneration, e.g. with acid, base or chaotropicagents, and suitable for heat sterilization, e.g. autoclaving at 120° C.for 20 min. The polymers are also useful for their ability to supportcell adherence and growth. The properties derived from dragline silk areattractive in development of new materials for medical or technicalpurposes. In particular, protein structures according to the inventionare useful in preparative and analytical separation procedures, such aschromatography, cell capture, selection and culture, active filters, anddiagnostics. Protein structures according to the invention are alsouseful in medical devices, such as implants and medical products, suchas wound closure systems, band-aids, sutures, wound dressings, andscaffolds for cell immobilization, cell culture, tissue engineering andguided cell regeneration.

The present invention provides a recombinant fusion protein that iscapable of selective interaction with an organic target, which fusionprotein is comprising the moieties B, REP and CT, and optionally NT. Thepresent invention also provides a protein structure that is capable ofselective interaction with an organic target, wherein said proteinstructure is a polymer comprising, and optionally consisting of, therecombinant fusion protein according to the invention, i.e. comprising,and optionally consisting of, the moieties B, REP and CT, and optionallyNT.

Although the REP and the CT moieties of the fusion proteins in theexamples by necessity relate to specific proteins, e.g. proteins derivedfrom major spidroin 1 (MaSp1) from Euprosthenops australis, it isconsidered that the present disclosure is applicable to any structurallysimilar moieties for the purpose of producing fusion protein structuresaccording to the invention. Furthermore, although the B moiety whichprovides the selective interaction activity of the fusion proteins inthe examples by necessity relate to specific protein moieties, e.g.moieties derived from protein A, protein G and streptavidin, it isconsidered that the present disclosure is applicable to any structurallyand/or functionally similar B moiety for the purpose of producing fusionprotein structures according to the invention, capable of selectiveinteraction with an organic target.

Specific fusion proteins according to the invention are defined by theformulas B_(x)—REP-B_(y)-CT-B_(z) and B_(z)—CT-B_(y)-REP-B_(z), whereinx, y and z are integers from 0 to 5; and x+y+z 1, optionally furthercontaining one NT moiety at either end of the fusion protein or betweenany two protein moieties in the fusion protein. If x+y+z>1, i.e. ifthere are two or more B moieties, they may be identical or different.The two or more B moieties may have capacity of selective interactionwith the same organic target or with different organic targets. It ispreferred that the two or more B moieties are substantially identical,each having capacity of selective interaction with the same organictarget.

In preferred fusion proteins according to the invention, x, y and z areintegers from 0 to 2, preferably from 0 to 1. In certain preferredfusion proteins according to the invention, y=0. In more preferredspecific fusion proteins according to the invention, y=0 and either x orz are 0, i.e. the fusion proteins are defined by the formulasB_(z)—REP-CT, B_(z)-CT-REP, REP-CT-B_(Z) and CT-REP-B_(Z), wherein x andz are integers from 1 to 5. In preferred fusion proteins according tothe invention, y=0, x and z are integers from 0 to 1; and x+z=1. Thus,certain preferred fusion proteins according to the invention are definedby the formulas B-REP-CT, B-CT-REP, REP-CT-B and CT-REP-B. In preferredfusion proteins according to the invention, the optional NT moiety ismissing.

The term “fusion protein” implies here a protein that is made byexpression from a recombinant nucleic acid, i.e. DNA or RNA that iscreated artificially by combining two or more nucleic acid sequencesthat would not normally occur together (genetic engineering). The fusionproteins according to the invention are recombinant proteins, and theyare therefore not identical to naturally occurring proteins. Inparticular, wildtype spidroins are not fusion proteins according to theinvention, because they are not expressed from a recombinant nucleicacid as set out above. The combined nucleic acid sequences encodedifferent proteins, partial proteins or polypeptides with certainfunctional properties. The resulting fusion protein, or recombinantfusion protein, is a single protein with functional properties derivedfrom each of the original proteins, partial proteins or polypeptides.Furthermore, the fusion protein according to the invention and thecorresponding genes are chimeric, i.e. the protein/gene moieties arederived from at least two different species. The REP and the CTmoieties, as well as the optional NT moiety, are all derived from aspider silk protein. For avoidance of doubt, the B moiety according tothe invention is a non-spidroin protein or polypeptide, i.e. it is notderived from a spider silk protein. In particular, the B moietyaccording to the invention is not derived from the C-terminal,repetitive or N-terminal fragments of a spider silk protein.

The fusion protein typically consists of from 170 to 2000 amino acidresidues, such as from 170 to 1000 amino acid residues, such as from 170to 600 amino acid residues, preferably from 170 to 500 amino acidresidues, such as from 170 to 400 amino acid residues. The small size isadvantageous because longer proteins containing spider silk proteinfragments may form amorphous aggregates, which require use of harshsolvents for solubilisation and polymerisation. The recombinant fusionprotein may contain more than 2000 residues, in particular in caseswhere the spider silk protein more than one B moiety and/or when itcontains a NT moiety.

The terms “spidroins” and “spider silk proteins” are usedinterchangeably throughout the description and encompass all knownspider silk proteins, including major ampullate spider silk proteinswhich typically are abbreviated “MaSp”, or “ADF” in the case of Araneusdiadematus. These major ampullate spider silk proteins are generally oftwo types, 1 and 2. These terms furthermore include non-natural proteinswith a high degree of identity and/or similarity to the known spidersilk proteins.

Consequently, the term “non-spidroin” implies proteins that are notderived from a spider silk protein, i.e. with a low (or no) degree ofidentity and/or similarity to spider silk proteins.

The protein structure according to the invention is capable of selectiveinteraction with an organic target. This capacity resides in the fusionprotein according to the invention, and more specifically in the Bmoiety of the fusion protein. Any interactions of the REP and the CTmoieties, as well as the optional NT moiety, with organic molecules arenot encompassed by the term “capable of selective interaction with anorganic target”. For avoidance of doubt, the term “capable of selectiveinteraction with an organic target” does not encompass dimerization,oligomerization or polymerization of the fusion proteins according tothe invention that rely on interactions involving the REP and the CTmoieties, as well as the optional NT moiety.

The term “organic target” encompasses all chemical molecules containingcarbon with the exception of what is traditionally considered inorganicmolecules by the skilled person, e.g. carbonates, simple oxides ofcarbon, cyanides, diamond and graphite. For avoidance of doubt,inorganic molecules, salts and ions, such as silica and calciumchloride, are not organic. The organic target may be a complexcontaining or consisting of organic molecules, e.g. a receptor complexon a cell surface. The organic target may be a monomer, dimer, oligomeror polymer of one or more organic molecule types, which may be heldtogether by covalent bonds or other types of association. It may ofcourse also simply be a single organic molecule. Preferred organictargets according to the invention include, but are not limited to,nucleic acids, proteins and polypeptides, lipids and carbohydrates, aswell as combinations thereof. Further preferred organic targetsaccording to the invention include, but are not limited to,immunoglobulins, molecules comprising immunoglobulin or derivativesthereof, albumin, molecules comprising albumin or derivatives thereof,biotin, and molecules comprising biotin or derivatives or analoguesthereof.

In the context of the present invention, “specific” or “selective”interaction of a ligand, e.g. a B moiety of the fusion protein accordingto the invention with its target means that the interaction is such thata distinction between specific and non-specific, or between selectiveand non-selective, interaction becomes meaningful. The interactionbetween two proteins is sometimes measured by the dissociation constant.The dissociation constant describes the strength of binding (oraffinity) between two molecules. Typically the dissociation constantbetween an antibody and its antigen is from 10⁻⁷ to 10⁻¹¹ M. However,high specificity does not necessarily require high affinity. Moleculeswith low affinity (in the molar range) for its counterpart have beenshown to be as specific as molecules with much higher affinity. In thecase of the present invention, a specific or selective interactionrefers to the extent to which a particular method can be used todetermine the presence and/or amount of a specific protein, the targetprotein or a fragment thereof, under given conditions in the presence ofother proteins in a sample of a naturally occurring or processedbiological or biochemical fluid. In other words, specificity orselectivity is the capacity to distinguish between related proteins.Specific and selective are sometimes used interchangeably in the presentdescription.

The fusion protein according to the invention may also contain one ormore linker peptides. The linker peptide(s) may be arranged between anymoieties of the fusion protein, e.g. between the CT and REP moieties,between two B moieties, between B and CT moieties, and between B and REPmoieties, or may be arranged at either terminal end of the fusionprotein. If the fusion protein contains two or more B moieties, thelinker peptide(s) may also be arranged in between two B moieties. Thelinker(s) may provide a spacer between the functional units of thefusion protein, but may also constitute a handle for identification andpurification of the fusion protein, e.g. a His and/or a Trx tag. If thefusion protein contains two or more linker peptides for identificationand purification of the fusion protein, it is preferred that they areseparated by a spacer sequence, e.g. His₆-spacer-His₆-. The linker mayalso constitute a signal peptide, such as a signal recognition particle,which directs the fusion protein to the membrane and/or causes secretionof the fusion protein from the host cell into the surrounding medium.The fusion protein may also include a cleavage site in its amino acidsequence, which allows for cleavage and removal of the linker(s) and/orother relevant moieties, typically the B moiety or moieties. Variouscleavage sites are known to the person skilled in the art, e.g. cleavagesites for chemical agents, such as CNBr after Met residues andhydroxylamine between Asn-Gly residues, cleavage sites for proteases,such as thrombin or protease 3C, and self-splicing sequences, such asintein self-splicing sequences.

The REP, CT and B moieties are linked directly or indirectly to oneanother. A direct linkage implies a direct covalent binding between themoieties without intervening sequences, such as linkers. An indirectlinkage also implies that the moieties are linked by covalent bonds, butthat there are intervening sequences, such as linkers and/or one or morefurther moieties, e.g. a NT moiety.

The B moiety or moieties may be arranged internally or at either end ofthe fusion protein, i.e. C-terminally arranged or N-terminally arranged.It is preferred that the B moiety or moieties are arranged at theN-terminal end of the fusion protein. If the fusion protein contains oneor more linker peptide(s) for identification and purification of thefusion protein, e.g. a His or Trx tag(s), it is preferred that it isarranged at the N-terminal end of the fusion protein.

A preferred fusion protein has the form of an N-terminally arranged Bmoiety, coupled by a linker peptide of 1-30 amino acid residues, such as1-10 amino acid residues, to C-terminally arranged REP and CT moieties.The linker peptide may contain a cleavage site. Optionally, the fusionprotein has an N-terminal or C-terminal linker peptide, which maycontain a purification tag, such as a His tag, and a cleavage site.

Another preferred fusion protein has the form of an N-terminallyarranged B moiety coupled directly to C-terminally arranged REP and CTmoieties. Optionally, the fusion protein has an N-terminal or C-terminallinker peptide, which may contain a purification tag, such as a His tag,and a cleavage site.

The protein structure according to the invention is a polymer comprisingas a repeating structural unit recombinant fusion proteins according tothe invention, which implies that it contains an ordered plurality offusion proteins according to the invention, typically well above 100fusion protein units, e.g. 1000 fusion protein units or more.Optionally, the polymer may comprise as a further repeating structuralunit complementary proteins without a B moiety, preferably proteinsderived from spider silk. This may be advantageous if the B moiety ofthe fusion protein is large and/or bulky. These complementary proteinstypically comprise a REP moiety and a CT moiety, and optionally an NTmoiety. Preferred complementary proteins according to the invention canhave any of the structures set out herein with a deleted B moiety. It ispreferred that the complementary fusion protein in is substantiallyidentical to the fusion protein with a deleted B moiety. However, it ispreferred that the protein structure according to the invention is apolymer consisting of recombinant fusion proteins according to theinvention as a repeating structural unit, i.e. that the proteinstructure according to the invention is a polymer of the recombinantfusion protein according to the invention.

The magnitude of fusion units in the polymer implies that the proteinstructure obtains a significant size. In a preferred embodiment, theprotein structure has a size of at least 0.1 μm in at least twodimensions. Thus, the term “protein structure” as used herein relates tofusion protein polymers having a thickness of at least 0.1 μm,preferably macroscopic polymers that are visible to the human eye, i.e.having a thickness of at least 1 μm. The term “protein structure” doesnot encompass unstructured aggregates or precipitates. While monomers ofthe fusion protein are water soluble, it is understood that the proteinstructures according to the invention are solid structures, i.e. notsoluble in water. The protein structures are polymers comprising as arepeating structural unit monomers of the recombinant fusion proteinsaccording to the invention.

It is preferable that the protein structure according to the inventionis in a physical form selected from the group consisting of fiber, film,foam, net, mesh, sphere and capsule.

It is preferable that the protein structure according to the inventionis a fiber or film with a thickness of at least 0.1 μm, preferably atleast 1 μm. It is preferred that the fiber or film has a thickness inthe range of 1-400 μm, preferably 60-120 μm. It is preferred that fibershave a length in the range of 0.5-300 cm, preferably 1-100 cm. Otherpreferred ranges are 0.5-30 cm and 1-20 cm. The fiber has the capacityto remain intact during physical manipulation, i.e. can be used forspinning, weaving, twisting, crocheting and similar procedures. The filmis advantageous in that it is coherent and adheres to solid structures,e.g. the plastics in microtiter plates. This property of the filmfacilitates washing and regeneration procedures and is very useful forseparation purposes. A particularly useful protein structure is a filmor a fiber wherein the B moiety is the Z domain derived fromstaphylococcal protein A or a protein fragment having at least 70%identity thereto, see e.g. Examples 1-6.

It is also preferred that the protein structure according to theinvention has a tensile strength above 1 MPa, preferably above 2 MPa,more preferably 10 MPa or higher. It is preferred that the proteinstructure according to the invention has a tensile strength above 100MPa, more preferably 200 MPa or higher.

The REP moiety is a protein fragment containing from 70 to 300 aminoacid residues and is derived from the repetitive fragment of a spidersilk protein. This implies that the REP moiety has a repetitivecharacter, alternating between alanine-rich stretches and glycine-richstretches. The REP moiety generally contains more than 70, such as morethan 140, and less than 300, preferably less than 240, such as less than200, amino acid residues, and can itself be divided into several L(linker) segments, A (alanine-rich) segments and G (glycine-rich)segments, as will be explained in more detail below. Typically, saidlinker segments, which are optional, are located at the REP moietyterminals, while the remaining segments are in turn alanine-rich andglycine-rich. Thus, the REP moiety can generally have either of thefollowing structures, wherein n is an integer:

L(AG)_(n)L, such as LA₁G₁A₂G₂A₃G₃A₄G₄A₅G₅L;

L(AG)_(n)AL, such as LA₁G₁A₂G₂A₃G₃A₄G₄A₅G₅A₆L;

L(GA)_(n)L, such as LG₁A₁G₂A₂G₃A₃G₄A₄G₅A₅L; or

L(GA)_(n)GL, such as LG₁A₁G₂A₂G₃A₃G₄A₄G₅A₅G₆L.

It follows that it is not critical whether an alanine-rich or aglycine-rich segment is adjacent to the N-terminal or C-terminal linkersegments. It is preferred that n is an integer from 2 to 10, preferablyfrom 2 to 8, preferably from 4 to 8, more preferred from 4 to 6, i.e.n=4, n=5 or n=6.

In preferred embodiments, the alanine content of the REP moietyaccording to the invention is above 20%, preferably above 25%, morepreferably above 30%, and below 50%, preferably below 40%, morepreferably below 35%. This is advantageous, since it is contemplatedthat a higher alanine content provides a stiffer and/or stronger and/orless extendible structure.

In certain embodiments, the REP moiety is void of proline residues, i.e.there are no proline residues in the REP moiety.

Now turning to the segments that constitute the REP moiety according tothe invention, it shall be emphasized that each segment is individual,i.e. any two A segments, any two G segments or any two L segments of aspecific REP moiety may be identical or may not be identical. Thus, itis not a general feature of the invention that each type of segment isidentical within a specific REP moiety. Rather, the following disclosureprovides the skilled person with guidelines how to design individualsegments and gather them into a REP moiety which is thereby consideredto be derived from the repetitive fragment of a spider silk protein, andwhich constitutes a part of a functional fusion protein according to theinvention.

Each individual A segment is an amino acid sequence having from 8 to 18amino acid residues. It is preferred that each individual A segmentcontains from 13 to 15 amino acid residues. It is also possible that amajority, or more than two, of the A segments contain from 13 to 15amino acid residues, and that a minority, such as one or two, of the Asegments contain from 8 to 18 amino acid residues, such as 8-12 or 16-18amino acid residues. A vast majority of these amino acid residues arealanine residues. More specifically, from 0 to 3 of the amino acidresidues are not alanine residues, and the remaining amino acid residuesare alanine residues. Thus, all amino acid residues in each individual Asegment are alanine residues, with no exception or the exception of one,two or three amino acid residues, which can be any amino acid. It ispreferred that the alanine-replacing amino acid(s) is (are) naturalamino acids, preferably individually selected from the group of serine,glutamic acid, cysteine and glycine, more preferably serine. Of course,it is possible that one or more of the A segments are all-alaninesegments, while the remaining A segments contain 1-3 non-alanineresidues, such as serine, glutamic acid, cysteine or glycine.

In a preferred embodiment, each A segment contains 13-15 amino acidresidues, including 10-15 alanine residues and 0-3 non-alanine residuesas described above. In a more preferred embodiment, each A segmentcontains 13-15 amino acid residues, including 12-15 alanine residues and0-1 non-alanine residues as described above.

It is preferred that each individual A segment has at least 80%,preferably at least 90%, more preferably 95%, most preferably 100%identity to an amino acid sequence selected from the group of amino acidresidues 7-19, 43-56, 71-83, 107-120, 135-147, 171-183, 198-211,235-248, 266-279, 294-306, 330-342, 357-370, 394-406, 421-434, 458-470,489-502, 517-529, 553-566, 581-594, 618-630, 648-661, 676-688, 712-725,740-752, 776-789, 804-816, 840-853, 868-880, 904-917, 932-945, 969-981,999-1013, 1028-1042 and 1060-1073 of SEQ ID NO: 10. Each sequence ofthis group corresponds to a segment of the naturally occurring sequenceof Euprosthenops australis MaSp1 protein, which is deduced from cloningof the corresponding cDNA, see WO 2007/078239. Alternatively, eachindividual A segment has at least 80%, preferably at least 90%, morepreferably 95%, most preferably 100% identity to an amino acid sequenceselected from the group of amino acid residues 143-152, 174-186,204-218, 233-247 and 265-278 of SEQ ID NO: 3. Each sequence of thisgroup corresponds to a segment of expressed, non-natural spider silkproteins, which proteins have capacity to form silk structures underappropriate conditions. Thus, in certain embodiments according to theinvention, each individual A segment is identical to an amino acidsequence selected from the above-mentioned amino acid segments. Withoutwishing to be bound by any particular theory, it is envisaged that Asegments according to the invention form helical structures or betasheets.

The term “% identity”, as used throughout the specification and theappended claims, is calculated as follows. The query sequence is alignedto the target sequence using the CLUSTAL W algorithm (Thompson, J. D.,Higgins, D. G. and Gibson, T. J., Nucleic Acids Research, 22: 4673-4680(1994)). A comparison is made over the window corresponding to theshortest of the aligned sequences. The amino acid residues at eachposition are compared, and the percentage of positions in the querysequence that have identical correspondences in the target sequence isreported as % identity.

The term “% similarity”, as used throughout the specification and theappended claims, is calculated as described for “% identity”, with theexception that the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile,Trp, Met and Cys are similar; the basic residues Lys, Arg and His aresimilar; the acidic residues Glu and Asp are similar; and thehydrophilic, uncharged residues Gln, Asn, Ser, Thr and Tyr are similar.The remaining natural amino acid Gly is not similar to any other aminoacid in this context.

Throughout this description, alternative embodiments according to theinvention fulfill, instead of the specified percentage of identity, thecorresponding percentage of similarity. Other alternative embodimentsfulfill the specified percentage of identity as well as another, higherpercentage of similarity, selected from the group of preferredpercentages of identity for each sequence. For example, a sequence maybe 70% similar to another sequence; or it may be 70% identical toanother sequence; or it may be 70% identical and 90% similar to anothersequence.

Furthermore, it has been concluded from experimental data that eachindividual G segment is an amino acid sequence of from 12 to 30 aminoacid residues. It is preferred that each individual G segment consistsof from 14 to 23 amino acid residues. At least 40% of the amino acidresidues of each G segment are glycine residues. Typically the glycinecontent of each individual G segment is in the range of 40-60%.

It is preferred that each individual G segment has at least 80%,preferably at least 90%, more preferably 95%, most preferably 100%identity to an amino acid sequence selected from the group of amino acidresidues 20-42, 57-70, 84-106, 121-134, 148-170, 184-197, 212-234,249-265, 280-293, 307-329, 343-356, 371-393, 407-420, 435-457, 471-488,503-516, 530-552, 567-580, 595-617, 631-647, 662-675, 689-711, 726-739,753-775, 790-803, 817-839, 854-867, 881-903, 918-931, 946-968, 982-998,1014-1027, 1043-1059 and 1074-1092 of SEQ ID NO: 10. Each sequence ofthis group corresponds to a segment of the naturally occurring sequenceof Euprosthenops australis MaSp1 protein, which is deduced from cloningof the corresponding cDNA, see WO 2007/078239. Alternatively, eachindividual G segment has at least 80%, preferably at least 90%, morepreferably 95%, most preferably 100% identity to an amino acid sequenceselected from the group of amino acid residues 153-173, 187-203,219-232, 248-264 and 279-296 of SEQ ID NO: 3. Each sequence of thisgroup corresponds to a segment of expressed, non-natural spider silkproteins, which proteins have capacity to form silk structures underappropriate conditions. Thus, in certain embodiments according to theinvention, each individual G segment is identical to an amino acidsequence selected from the above-mentioned amino acid segments.

In certain embodiments, the first two amino acid residues of each Gsegment according to the invention are not -Gln-Gln-.

There are the three subtypes of the G segment according to theinvention. This classification is based upon careful analysis of theEuprosthenops australis MaSp1 protein sequence (WO 2007/078239), and theinformation has been employed and verified in the construction of novel,non-natural spider silk proteins.

The first subtype of the G segment according to the invention isrepresented by the amino acid one letter consensus sequenceGQG(G/S)QGG(Q/Y)GG (L/Q)GQGGYGQGA GSS (SEQ ID NO: 11). This first, andgenerally the longest, G segment subtype typically contains 23 aminoacid residues, but may contain as little as 17 amino acid residues, andlacks charged residues or contain one charged residue. Thus, it ispreferred that this first G segment subtype contains 17-23 amino acidresidues, but it is contemplated that it may contain as few as 12 or asmany as 30 amino acid residues. Without wishing to be bound by anyparticular theory, it is envisaged that this subtype forms coilstructures or 3₁-helix structures. Representative G segments of thisfirst subtype are amino acid residues 20-42, 84-106, 148-170, 212-234,307-329, 371-393, 435-457, 530-552, 595-617, 689-711, 753-775, 817-839,881-903, 946-968, 1043-1059 and 1074-1092 of SEQ ID NO: 10. In certainembodiments, the first two amino acid residues of each G segment of thisfirst subtype according to the invention are not -Gln-Gln-.

The second subtype of the G segment according to the invention isrepresented by the amino acid one letter consensus sequenceGQGGQGQG(G/R)Y GQG(A/S)G(S/G)S (SEQ ID NO: 12). This second, generallymid-sized, G segment subtype typically contains 17 amino acid residuesand lacks charged residues or contain one charged residue. It ispreferred that this second G segment subtype contains 14-20 amino acidresidues, but it is contemplated that it may contain as few as 12 or asmany as 30 amino acid residues. Without wishing to be bound by anyparticular theory, it is envisaged that this subtype forms coilstructures. Representative G segments of this second subtype are aminoacid residues 249-265, 471-488, 631-647 and 982-998 of SEQ ID NO: 10;and amino acid residues 187-203 of SEQ ID NO: 3.

The third subtype of the G segment according to the invention isrepresented by the amino acid one letter consensus sequenceG(R/Q)GQG(G/R)YGQG (A/S/V)GGN (SEQ ID NO: 13). This third G segmentsubtype typically contains 14 amino acid residues, and is generally theshortest of the G segment subtypes according to the invention. It ispreferred that this third G segment subtype contains 12-17 amino acidresidues, but it is contemplated that it may contain as many as 23 aminoacid residues. Without wishing to be bound by any particular theory, itis envisaged that this subtype forms turn structures. Representative Gsegments of this third subtype are amino acid residues 57-70, 121-134,184-197, 280-293, 343-356, 407-420, 503-516, 567-580, 662-675, 726-739,790-803, 854-867, 918-931, 1014-1027 of SEQ ID NO: 10; and amino acidresidues 219-232 of SEQ ID NO: 3.

Thus, in preferred embodiments, each individual G segment has at least80%, preferably 90%, more preferably 95%, identity to an amino acidsequence selected from SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

In a preferred embodiment of the alternating sequence of A and Gsegments of the REP moiety, every second G segment is of the firstsubtype, while the remaining G segments are of the third subtype, e.g. .. . A₁G_(short)A₂G_(long)A₃G_(short)A₄G_(long)A₅G_(short) . . . . Inanother preferred embodiment of the REP moiety, one G segment of thesecond subtype interrupts the G segment regularity via an insertion,e.g. . . . A₁G_(short)A₂G_(long)A₃G_(mid)A₄G_(short)A₅G_(long) . . . .

Each individual L segment represents an optional linker amino acidsequence, which may contain from 0 to 20 amino acid residues, such asfrom 0 to 10 amino acid residues. While this segment is optional and notfunctionally critical for the spider silk protein, its presence stillallows for fully functional spider silk fusion proteins, forming proteinstructures according to the invention. There are also linker amino acidsequences present in the repetitive part (SEQ ID NO: 10) of the deducedamino acid sequence of the MaSp1 protein from Euprosthenops australis.In particular, the amino acid sequence of a linker segment may resembleany of the described A or G segments, but usually not sufficiently tomeet their criteria as defined herein.

As shown in WO 2007/078239, a linker segment arranged at the C-terminalpart of the REP moiety can be represented by the amino acid one letterconsensus sequences ASASAAASAA STVANSVS and ASAASAAA, which are rich inalanine. In fact, the second sequence can be considered to be an Asegment according to the invention, while the first sequence has a highdegree of similarity to A segments according to the invention. Anotherexample of a linker segment according the invention has the one letteramino acid sequence GSAMGQGS, which is rich in glycine and has a highdegree of similarity to G segments according to the invention. Anotherexample of a linker segment is SASAG.

Representative L segments are amino acid residues 1-6 and 1093-1110 ofSEQ ID NO: 10; and amino acid residues 138-142 of SEQ ID NO: 3, but theskilled person in the art will readily recognize that there are manysuitable alternative amino acid sequences for these segments. In oneembodiment of the REP moiety according to the invention, one of the Lsegments contains 0 amino acids, i.e. one of the L segments is void. Inanother embodiment of the REP moiety according to the invention, both Lsegments contain 0 amino acids, i.e. both L segments are void. Thus,these embodiments of the REP moieties according to the invention may beschematically represented as follows: (AG)_(n)L, (AG)_(n)AL, (GA)_(n)L,(GA)_(n)GL; L(AG)_(n), L(AG)_(n)A, L(GA)_(n), L(GA)_(n)G; and (AG)_(n),(AG)_(n)A, (GA)_(n), (GA)_(n)G. Any of these REP moieties are suitablefor use with any CT moiety as defined below.

The CT moiety is a protein fragment containing from 70 to 120 amino acidresidues and is derived from the C-terminal fragment of a spider silkprotein. The expression “derived from” implies in the context of the CTmoiety according to the invention that it has a high degree ofsimilarity to the C-terminal amino acid sequence of spider silkproteins. As shown in FIG. 1, this amino acid sequence is well conservedamong various species and spider silk proteins, including MaSp1 andMaSp2. A consensus sequence of the C-terminal regions of MaSp1 and MaSp2is provided as SEQ ID NO: 9. In FIG. 1, the following MaSp proteins arealigned, denoted with GenBank accession entries where applicable:

TABLE 1 Spidroin CT moieties Species and spidroin protein EntryEuprosthenops sp MaSp1 (Pouchkina-Stantcheva, Cthyb_Esp N N &McQueen-Mason, S J, ibid) Euprosthenops australis MaSp1 CTnat_EauArgiope trifasciata MaSp1 AF350266_At1 Cyrtophora moluccensis Sp1AY666062_Cm1 Latrodectus geometricus MaSp1 AF350273_Lg1 Latrodectushesperus MaSp1 AY953074_Lh1 Macrothele holsti Sp1 AY666068_Mh1 Nephilaclavipes MaSp1 U20329_Nc1 Nephila pilipes MaSp1 AY666076_Np1 Nephilamadagascariensis MaSp1 AF350277_Nm1 Nephila senegalensis MaSp1AF350279_Ns1 Octonoba varians Sp1 AY666057_Ov1 Psechrus sinensis Sp1AY666064_Ps1 Tetragnatha kauaiensis MaSp1 AF350285_Tk1 Tetragnathaversicolor MaSp1 AF350286_Tv1 Araneus bicentenarius Sp2 ABU20328_Ab2Argiope amoena MaSp2 AY365016_Aam2 Argiope aurantia MaSp2 AF350263_Aau2Argiope trifasciata MaSp2 AF350267_At2 Gasteracantha mammosa MaSp2AF350272_Gm2 Latrodectus geometricus MaSp2 AF350275_Lg2 Latrodectushesperus MaSp2 AY953075_Lh2 Nephila clavipes MaSp2 AY654293_Nc2 Nephilamadagascariensis MaSp2 AF350278_Nm2 Nephila senegalensis MaSp2AF350280_Ns2 Dolomedes tenebrosus Fb1 AF350269_DtFb1 Dolomedestenebrosus Fb2 AF350270_DtFb2 Araneus diadematus ADF-1 U47853_ADF1Araneus diadematus ADF-2 U47854_ADF2 Araneus diadematus ADF-3U47855_ADF3 Araneus diadematus ADF-4 U47856_ADF4

It is not critical which specific CT moiety is present in spider silkproteins according to the invention, as long as the CT moiety is notentirely missing. Thus, the CT moiety according to the invention can beselected from any of the amino acid sequences shown in FIG. 1 and Table1 or sequences with a high degree of similarity. A wide variety ofC-terminal sequences can be used in the spider silk protein according tothe invention.

The sequence of the CT moiety according to the invention has at least50% identity, preferably at least 60%, more preferably at least 65%identity, or even at least 70% identity, to the consensus amino acidsequence SEQ ID NO: 9, which is based on the amino acid sequences ofFIG. 1.

A representative CT moiety according to the invention is theEuprosthenops australis sequence SEQ ID NO: 7, Thus, according to apreferred aspect of the invention, the CT moiety has at least 80%,preferably at least 90%, such as at least 95%, identity to SEQ ID NO: 7or any individual amino acid sequence of FIG. 1 and Table 1. Inpreferred aspects of the invention, the CT moiety is identical to SEQ IDNO: 7 or any individual amino acid sequence of FIG. 1 and Table 1.

The CT moiety typically consists of from 70 to 120 amino acid residues.It is preferred that the CT moiety contains at least 70, or more than80, preferably more than 90, amino acid residues. It is also preferredthat the CT moiety contains at most 120, or less than 110 amino acidresidues. A typical CT moiety contains approximately 100 amino acidresidues.

The optional NT moiety is a protein fragment containing from 100 to 160amino acid residues and is derived from the N-terminal fragment of aspider silk protein. The expression “derived from” implies in thecontext of the NT moiety according to the invention that it has a highdegree of similarity to the N-terminal amino acid sequence of spidersilk proteins. As shown in FIG. 2, this amino acid sequence is wellconserved among various species and spider silk proteins, includingMaSp1 and MaSp2. In FIG. 2, the following spidroin NT moieties arealigned, denoted with Gen Bank accession entries where applicable:

TABLE 2 Spidroin NT moieties GenBank Code Species and spidroin proteinacc. no. Ea MaSp1 Euprosthenops australis MaSp 1 AM259067 Lg MaSp1Latrodectus geometricus MaSp 1 ABY67420 Lh MaSp1 Latrodectus hesperusMaSp 1 ABY67414 Nc MaSp1 Nephila clavipes MaSp 1 ACF19411 At MaSp2Argiope trifasciata MaSp 2 AAZ15371 Lg MaSp2 Latrodectus geometricusMaSp 2 ABY67417 Lh MaSp2 Latrodectus hesperus MaSp 2 ABR68855 Nim MaSp2Nephila inaurata madagascariensis AAZ15322 MaSp 2 Nc MaSp2 Nephilaclavipes MaSp 2 ACF19413 Ab CySp1 Argiope bruennichi cylindriformspidroin 1 BAE86855 Ncl CySp1 Nephila clavata cylindriform spidroin 1BAE54451 Lh TuSp1 Latrodectus hesperus tubuliform spidroin ABD24296 NcFlag Nephila clavipes flagelliform silk protein AF027972 Nim FlagNephila inaurata madagascariensis AF218623 flagelliform silk protein(translated)

Only the part corresponding to the N-terminal moiety is shown for eachsequence, omitting the signal peptide. Nc flag and Nlm flag aretranslated and edited according to Rising A. et al. Biomacromolecules 7,3120-3124 (2006)).

It is not critical which specific NT moiety is present in spider silkproteins according to the invention. Thus, the NT moiety according tothe invention can be selected from any of the amino acid sequences shownin FIG. 2 or sequences with a high degree of similarity. A wide varietyof N-terminal sequences can be used in the spider silk protein accordingto the invention. Based on the homologous sequences of FIG. 2, thefollowing sequence constitutes a consensus NT amino acid sequence:

(SEQ ID NO: 8) QANTPWSSPNLADAFINSF(M/L)SA(A/I)SSSGAFSADQLDDMSTIG(D/N/Q)TLMSAMD(N/S/K)MGRSG(K/R)STKSKLQALNMAFASSMAEIAAAESGG(G/Q)SVGVKTNAISDALSSAFYQTTGSVNPQFV(N/S)EIRSLI(G/N)M(F/L)(A/S)QASANEV.

The sequence of the NT moiety according to the invention has at least50% identity, preferably at least 60% identity, to the consensus aminoacid sequence SEQ ID NO: 8, which is based on the amino acid sequencesof FIG. 2. In a preferred embodiment, the sequence of the NT moietyaccording to the invention has at least 65% identity, preferably atleast 70% identity, to the consensus amino acid sequence SEQ ID NO: 8.In preferred embodiments, the NT moiety according to the invention hasfurthermore 70%, preferably 80%, similarity to the consensus amino acidsequence SEQ ID NO: 8.

A representative NT moiety according to the invention is theEuprosthenops australis sequence SEQ ID NO: 6. According to a preferredembodiment of the invention, the NT moiety has at least 80% identity toSEQ ID NO: 6 or any individual amino acid sequence in FIG. 1. Inpreferred embodiments of the invention, the NT moiety has at least 90%,such as at least 95% identity, to SEQ ID NO: 6 or any individual aminoacid sequence in FIG. 2. In preferred embodiments of the invention, theNT moiety is identical to SEQ ID NO: 6 or any individual amino acidsequence in FIG. 1, in particular to Ea MaSp1.

The NT moiety contains from 100 to 160 amino acid residues. It ispreferred that the NT moiety contains at least 100, or more than 110,preferably more than 120, amino acid residues. It is also preferred thatthe NT moiety contains at most 160, or less than 140 amino acidresidues. A typical NT moiety contains approximately 130-140 amino acidresidues.

The B moiety is a protein or polypeptide fragment comprising more than30 amino acid residues. The B moiety is preferably comprising more than40 amino acid residues, such as more than 50 amino acid residues. The Bmoiety is preferably comprising less than 500 amino acid residues, suchas less than 200 amino acid residues, more preferably less than 100amino acid residues, such as less than 100 amino acid residues. It iscapable of selective interaction with the organic target, and it is theB moiety in the fusion protein which provides the capacity of selectiveinteraction with the organic target.

The B moiety is a non-spidroin moiety. This implies that it is notderived from a spider silk protein, i.e. it has a low (or no) degree ofidentity and/or similarity to spider silk proteins. The sequence of theB moiety according to the invention preferably has less than 30%identity, such as less than 20% identity, preferably less than 10%identity, to any of the spidroin amino acid sequences disclosed herein,and specifically to any of SEQ ID NO: 6-10.

It is regarded as within the capabilities of those of ordinary skill inthe art to select the B moiety. Nevertheless, examples of affinityligands that may prove useful as B moieties, as well as examples offormats and conditions for detection and/or quantification, are givenbelow for the sake of illustration.

The biomolecular diversity needed for selection of affinity ligands maybe generated by combinatorial engineering of one of a plurality ofpossible scaffold molecules, and specific and/or selective affinityligands are then selected using a suitable selection platform.Non-limiting examples of such structures, useful for generating affinityligands against the organic target, are staphylococcal protein A anddomains thereof and derivatives of these domains, such as the Z domain(Nord K et al. (1997) Nat. Biotechnol. 15:772-777); lipocalins (Beste Get al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:1898-1903); ankyrinrepeat domains (Binz H K et al. (2003) J. Mol. Biol. 332:489-503);cellulose binding domains (CBD) (Smith G P et al. (1998) J. Mol. Biol.277:317-332; Lehtiö J et al. (2000) Proteins 41:316-322); γ crystallines(Fiedler U and Rudolph R, WO01/04144); green fluorescent protein (GFP)(Peelle B et al. (2001) Chem. Biol. 8:521-534); human cytotoxic Tlymphocyte-associated antigen 4 (CTLA-4) (Hufton S E et al. (2000) FEBSLett. 475:225-231; Irving R A et al. (2001) J. Immunol. Meth.248:31-45); protease inhibitors, such as Knottin proteins (Wentzel A etal. (2001) J. Bacteriol. 183:7273-7284; Baggio R et al. (2002) J. Mol.Recognit. 15:126-134) and Kunitz domains (Roberts B L et al. (1992) Gene121:9-15; Dennis M S and Lazarus R A (1994) J. Biol. Chem.269:22137-22144); PDZ domains (Schneider S et al. (1999) Nat.Biotechnol. 17:170-175); peptide aptamers, such as thioredoxin (Lu Z etal. (1995) Biotechnology 13:366-372; Klevenz B et al. (2002) Cell. Mol.Life Sci. 59:1993-1998); staphylococcal nuclease (Norman T C et al.(1999) Science 285:591-595); tendamistats (McConell S J and Hoess R H(1995) J. Mol. Biol. 250:460-479; Li R et al. (2003) Protein Eng.16:65-72); trinectins based on the fibronectin type III domain (Koide Aet al. (1998) J. Mol. Biol. 284:1141-1151; Xu L et al. (2002) Chem.Biol. 9:933-942); and zinc fingers (Bianchi E et al. (1995) J. Mol.Biol. 247:154-160; Klug A (1999) J. Mol. Biol. 293:215-218; Segal D J etal. (2003) Biochemistry 42:2137-2148).

The above-mentioned examples include scaffold proteins presenting asingle randomized loop used for the generation of novel bindingspecificities, protein scaffolds with a rigid secondary structure whereside chains protruding from the protein surface are randomized for thegeneration of novel binding specificities, and scaffolds exhibiting anon-contiguous hyper-variable loop region used for the generation ofnovel binding specificities.

Oligonucleotides may also be used as affinity ligands. Single strandednucleic acids, called aptamers or decoys, fold into well-definedthree-dimensional structures and bind to their target with high affinityand specificity. (Ellington A D and Szostak J W (1990) Nature346:818-822; Brody E N and Gold L (2000) J. Biotechnol. 74:5-13; Mayer Gand Jenne A (2004) BioDrugs 18:351-359). The oligonucleotide ligands canbe either RNA or DNA and can bind to a wide range of target moleculeclasses.

For selection of the desired affinity ligand from a pool of variants ofany of the scaffold structures mentioned above, a number of selectionplatforms are available for the isolation of a specific novel ligandagainst a target protein of choice. Selection platforms include, but arenot limited to, phage display (Smith G P (1985) Science 228:1315-1317),ribosome display (Hanes J and Plückthun A (1997) Proc. Natl. Acad. Sci.U.S.A. 94:4937-4942), yeast two-hybrid system (Fields S and Song 0(1989) Nature 340:245-246), yeast display (Gai S A and Wittrup K D(2007) Curr Opin Struct Biol 17:467-473), mRNA display (Roberts R W andSzostak J W (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12297-12302),bacterial display (Daugherty P S (2007) Curr Opin Struct Biol17:474-480, Kronqvist N et al. (2008) Protein Eng Des Sel 1-9, Harvey BR et al. (2004) PNAS 101(25):913-9198), microbead display (Nord O et al.(2003) J Biotechnol 106:1-13, WO01/05808), SELEX (System Evolution ofLigands by Exponential Enrichment) (Tuerk C and Gold L (1990) Science249:505-510) and protein fragment complementation assays (PCA) (Remy Iand Michnick S W (1999) Proc. Natl. Acad. Sci. U.S.A. 96:5394-5399). Apreferred group of B moieties with affinity for immunoglobulins, albuminor other organic targets are bacterial receptin domains or derivativesthereof.

A group of preferred B moieties are capable of selective interactionwith immunoglobulins and molecules comprising immunoglobulin orderivatives thereof. A preferred group of immunoglobulin subclasses arethe subclasses that are recognized by the Z domain derived fromstaphylococcal protein A, i.e. IgG1, IgG2, IgG4, IgA and IgM from human,all Ig subclasess from rabbit and cow, IgG1 and IgG2 from guinea pig,and IgG1, IgG2a, IgG2b, IgG3 and IgM from mouse (see Hober, S. et al.,J. Chromatogr B. 848:40-47 (2007)), more preferably the immunoglobulinsubclasses IgG1, IgG2, IgG4, IgA and IgM from human. Another preferredgroup of immunoglobulin subclasses are the subclasses that arerecognized by the C2 domain streptococcal protein G; i.e. all humansubclasses of IgG, including IgG3, and IgG from several animals,including mouse, rabbit and sheep.

One group of preferred B moieties are selected from the group consistingof the Z domain derived from staphylococcal protein A, staphylococcalprotein A and domains thereof, preferably the E, D, A, B and C domains,streptococcal protein G and domains thereof, preferably the C1, C2 andC3 domains; and protein fragments having at least 70% identity, such asat least 80% identity, or at least 90% identity, to any of these aminoacid sequences. Preferably, the B moiety is selected from the groupconsisting of the Z domain derived from staphylococcal protein A, the Bdomain of staphylococcal protein A, and the C2 domain of streptococcalprotein G; and protein fragments having at least 70% identity, such asat least 80% identity, or at least 90% identity, to any of these aminoacid sequences. Preferably, the B moiety is selected from the groupconsisting of the Z domain derived from staphylococcal protein A andprotein fragments having at least 70% identity, such as at least 80%identity, or at least 90% identity, to this amino acid sequence. It ispreferred that the B moiety is selected from the group consisting of theZ domain derived from staphylococcal protein A and the C2 domain ofstreptococcal protein G, see e.g. Examples 1-6 and 8. A preferred groupof B moieties with affinity for immunoglobulins are bacterial receptindomains or derivatives thereof.

Another group of preferred B moieties are capable of selectiveinteraction with albumin and molecules comprising albumin or derivativesthereof. A preferred group of B moieties with affinity for albumin arebacterial receptin domains or derivatives thereof. Preferred B moietiesare selected from streptococcal protein G, the albumin-binding domain ofstreptococcal protein G, GA modules from Finegoldia magna; and proteinfragments having at least 70% identity, such as at least 80% identity,or at least 90% identity, to any of these amino acid sequences.Preferably, the B moiety is selected from the albumin-binding domain ofstreptococcal protein G and protein fragments having at least 70%identity, such as at least 80% identity, or at least 90% identity,thereto. It is preferred that the B moiety is the albumin-binding domainof streptococcal protein G see e.g. Example 7.

A further group of preferred B moieties are capable of selectiveinteraction with biotin and molecules comprising biotin or derivativesor analogues thereof. Preferred B moieties are selected from the groupconsisting of streptavidin, monomeric streptavidin (M4); and proteinfragments having at least 70% identity, such as at least 80% identity,or at least 90% identity to any of these amino acid sequences. It ispreferred that the B moiety is monomeric streptavidin (M4) see e.g.Examples 10-12.

Specific fusion proteins and protein structures according to theinvention are provided in the Examples. These preferred fusion proteinsform the group consisting of SEQ ID NOS 14, 16, 18, 22, 24 and 26.Further preferred fusion proteins are having at least 80%, preferably atleast 90%, more preferably at least 95%, identity to any of thesesequences.

The present invention further provides isolated polynucleic acidsencoding a fusion protein according to the invention. In particular,specific polynucleic acids are provided in the Examples and the appendedsequence listing, e.g. SEQ ID NOS 15, 17, 19, 23, 25 and 27. Furtherpreferred polynucleic acids encode fusion proteins having at least 80%,preferably at least 90%, more preferably at least 95%, identity to anyof SEQ ID NOS 14, 16, 18, 22, 24 and 26.

The polynucleic acids according to the invention are useful forproducing the fusion proteins according to the invention. The presentinvention provides a method of producing a fusion protein. The firststep involves expressing in a suitable host a fusion protein accordingto the invention. Suitable hosts are well known to a person skilled inthe art and include e.g. bacteria and eukaryotic cells, such as yeast,insect cell lines and mammalian cell lines. Typically, this stepinvolves expression of a polynucleic acid molecule which encodes thefusion protein in E. coli.

The second method step involves obtaining a mixture containing thefusion protein. The mixture may for instance be obtained by lysing ormechanically disrupting the host cells. The mixture may also be obtainedby collecting the cell culture medium, if the fusion protein is secretedby the host cell. The thus obtained protein can be isolated usingstandard procedures. If desired, this mixture can be subjected tocentrifugation, and the appropriate fraction (precipitate orsupernatant) be collected. The mixture containing the fusion protein canalso be subjected to gel filtration, chromatography, e.g. anion exchangechromatography, dialysis, phase separation or filtration to causeseparation. Optionally, lipopolysaccharides and other pyrogens areactively removed at this stage. If desired, linker peptides may beremoved by cleavage in this step.

Proteins structures according to the invention are assembledspontaneously from the fusion proteins according to the invention undersuitable conditions, and the assembly into polymers is promoted by thepresence of shearing forces and/or an interface between two differentphases e.g. between a solid and a liquid phase, between air and a liquidphase or at a hydrophobic/hydrophilic interface, e.g. a mineraloil-water interface. The presence of the resulting interface stimulatespolymerization at the interface or in the region surrounding theinterface, which region extends into the liquid medium, such that saidpolymerizing initiates at said interface or in said interface region.Various protein structures can be produced by adapting the conditionsduring the assembly. For instance, if the assembly is allowed to occurin a container that is gently wagged from side to side, a fiber isformed at the air-water interface. If the mixture is allowed to standstill, a film is formed at the air-water interface. If the mixture isevaporated, a film is formed at the bottom of the container. If oil isadded on top of the aqueous mixture, a film is formed at the oil-waterinterface, either if allowed to stand still or if wagged. If the mixtureis foamed, e.g. by bubbling of air or whipping, the foam is stable andsolidifies if allowed to dry.

The present invention thus provides a method for providing a proteinstructure displaying a binding activity towards an organic target. Inthe first method step, there is provided a recombinant fusion proteinaccording to the invention. The fusion protein may e.g. be provided byexpressing it in a suitable host from a polynucleic acid according tothe invention. In the second method step, the fusion protein issubjected to conditions to achieve formation of a polymer comprising therecombinant fusion protein. Notably, although the spontaneouslyassembled protein structures can be solubilized inhexafluoroisopropanol, the solubilized fusion proteins are then not ableto spontaneously reassemble into e.g. fibers.

The protein structure is useful as part of an affinity medium forimmobilization of an organic target, wherein the B moiety is capable ofselective interaction with the organic target. A sample, e.g. abiological sample, may be applied to a fusion protein or a proteinstructure according to the invention which is capable of binding to anorganic target present in the biological sample, and the fusion proteinor protein structure is then useful in separation of the organic targetfrom the sample. A biological sample, such as blood, serum or plasmawhich has been removed from a subject may be subjected to detection,separation and/or quantification of the organic target.

The present invention thus provides a method for separation of anorganic target from a sample. A sample, e.g. a biological sample such asblood, serum or plasma, containing the organic target is provided. Thebiological sample may be an earlier obtained sample. If using an earlierobtained sample in a method, no steps of the method are practiced on thehuman or animal body.

An affinity medium according to the invention is provided, comprising afusion protein or a protein structure according to the invention. Incertain embodiments, the affinity medium is consisting of the fusionprotein or protein structure according to the invention. The affinitymedium is capable of selective interaction with the organic target bymeans of the B moiety in the fusion protein according to the invention.The affinity medium is contacted with the sample under suitableconditions to achieve binding between the affinity medium and theorganic target. Non-bound sample is removed under suitable conditions tomaintain selective binding between the affinity medium and the organictarget. This method results in an organic target immobilized to theaffinity medium, and specifically to the fusion protein, according tothe invention.

In a preferred method according to the invention, the fusion protein inthe affinity medium is present as a protein structure according to theinvention when contacting the affinity medium with the sample to achievebinding between the affinity medium and the organic target.

A particularly useful protein structure in this respect is a film or afiber wherein the B moiety is the Z domain derived from staphylococcalprotein A or a protein fragment having at least 70% identity, such as atleast 80% identity, or at least 90% identity, thereto, see e.g. Example1-6. The film is advantageous in that it adheres to solid structures,e.g. the plastics in microtiter plates. This property of the filmfacilitates washing and regeneration procedures and is very useful forseparation purposes.

It has surprisingly been observed that the alkali stability of the Zdomain may even be enhanced when being part of a fusion proteinaccording to the invention in a protein structure according to theinvention. This property may be very useful for washing and regenerationpurposes, e.g. allowing for high concentrations of NaOH, such as 0.1 M,0.5 M, 1 M or even above 1 M, e.g. 2 M, and/or for high concentrationsof urea, e.g. 6-8 M. The chemical stability may also be useful to allowfor repeated cycles of use of the Z domain for affinity purification.This alkali stability may be further increased by utilizing a stabilizedmutant of the Z domain. Furthermore, it has advantageously been shownthat the fusion proteins according to the invention, including the Zdomain, are heat stable. This allows for sterilization by heat withmaintained binding ability.

A known problem with traditional affinity matrices with Z domains isleakage of the Z domain from the affinity matrix. Due to the stableincorporation of the Z domain by a peptide bond into the fusion proteinof the invention, it is contemplated that the undesirable leakage of theZ domain from the protein structures according to the invention is lowor absent. Another advantage of the fusion proteins according to theinvention is that the resulting protein structure has a high density ofZ domains (or other B moieties). It is contemplated that this highdensity provides a high binding capacity. Altogether, these propertiesof the fusions proteins are very attractive for various B moieties, andin particular for affinity purification using protein Z with goodproduction economy. These properties are also useful in other formatsthan in traditional gel bead affinity columns, e.g. in filter-likeformats.

The immobilized organic target is capable of selective interaction witha second organic target. The method is then further comprising the stepof contacting said affinity medium and the immobilized organic targetwith a second organic target, which is capable of selective interactionwith the first organic target, under suitable conditions to achievebinding between the first and second organic targets.

The immobilized organic target is detectable and/or quantifiable. Thedetection and/or quantification of the organic target may beaccomplished in any way known to the skilled person for detection and/orquantification of binding reagents in assays based on various biologicalor non-biological interactions. The organic targets may be labeledthemselves with various markers or may in turn be detected by secondary,labeled affinity ligands to allow detection, visualization and/orquantification. This can be accomplished using any one or more of amultitude of labels, which can be conjugated to the organic target or toany secondary affinity ligand, using any one or more of a multitude oftechniques known to the skilled person, and not as such involving anyundue experimentation. Non-limiting examples of labels that can beconjugated to organic targets and/or secondary affinity ligands includefluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin,fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescentcompounds (e.g., luminal, imidazole) and bioluminescent proteins (e.g.,luciferin, luciferase), haptens (e.g., biotin). A variety of otheruseful fluorescers and chromophores are described in Stryer L (1968)Science 162:526-533 and Brand L and Gohlke J R (1972) Annu. Rev.Biochem. 41:843-868. Organic targets and/or secondary affinity ligandscan also be labeled with enzymes (e.g., horseradish peroxidase, alkalinephosphatase, beta-lactamase), radioisotopes (e.g., ³H, ¹⁴C, ³²P, ³⁵S or¹²⁵I) and particles (e.g., gold). In the context of the presentdisclosure, “particles” refer to particles, such as metal particles,suitable for labeling of molecules. Further, the affinity ligands mayalso be labeled with fluorescent semiconductor nanocrystals (quantumdots). Quantum dots have superior quantum yield and are more photostablecompared to organic fluorophores and are therefore more easily detected(Chan et al. (2002) Curr Opi Biotech. 13: 40-46). The different types oflabels can be conjugated to an organic target or a secondary affinityligand using various chemistries, e.g., the amine reaction or the thiolreaction. However, other reactive groups than amines and thiols can beused, e.g., aldehydes, carboxylic acids and glutamine.

If the detection and/or quantification involves exposure to a secondorganic target or secondary affinity ligand, the affinity medium iswashed once again with buffers to remove unbound secondary affinityligands. As an example, the secondary affinity ligand may be an antibodyor a fragment or a derivative thereof. Thereafter, organic targets maybe detected and/or quantified with conventional methods. The bindingproperties for a secondary affinity ligand may vary, but those skilledin the art should be able to determine operative and optimal assayconditions for each determination by routine experimentation.

The detection, localization and/or quantification of a labeled moleculemay involve visualizing techniques, such as light microscopy orimmunofluoresence microscopy. Other methods may involve the detectionvia flow cytometry or luminometry. The method of visualization of labelsmay include, but is not restricted to, fluorometric, luminometric and/orenzymatic techniques. Fluorescence is detected and/or quantified byexposing fluorescent labels to light of a specific wavelength andthereafter detecting and/or quantifying the emitted light in a specificwavelength region. The presence of a luminescently tagged molecule maybe detected and/or quantified by luminescence developed during achemical reaction. Detection of an enzymatic reaction is due to a colorshift in the sample arising from chemical reaction. Those of skill inthe art are aware that a variety of different protocols can be modifiedin order for proper detection and/or quantification.

One available method for detection and/or quantification of the organictarget is by linking it or the secondary affinity ligand to an enzymethat can then later be detected and/or quantified in an enzymeimmunoassay (such as an EIA or ELISA). Such techniques are wellestablished, and their realization does not present any unduedifficulties to the skilled person. In such methods, the biologicalsample is brought into contact with a protein structure according to theinvention which binds to the organic target, which is then detectedand/or quantified with an enzymatically labeled secondary affinityligand. Following this, an appropriate substrate is brought to react inappropriate buffers with the enzymatic label to produce a chemicalmoiety, which for example is detected and/or quantified using aspectrophotometer, fluorometer, luminometer or by visual means.

The organic target or the secondary affinity ligands can be labeled withradioisotopes to enable detection and/or quantification. Non-limitingexamples of appropriate radiolabels in the present disclosure are ³H,¹⁴C, ³²P, ³⁵S or ¹²⁵I. The specific activity of the labeled affinityligand is dependent upon the half-life of the radiolabel, isotopicpurity, and how the label has been incorporated into the affinityligand. Affinity ligands are preferably labeled using well-knowntechniques (Wensel T G and Meares C F (1983) in: Radioimmunoimaging andRadioimmunotherapy (Burchiel S W and Rhodes B A eds.) Elsevier, NewYork, pp 185-196). A thus radiolabeled affinity ligand can be used tovisualize the organic target by detection of radioactivity. Radionuclearscanning can be performed with e.g. a gamma camera, magnetic resonancespectroscopy, emission tomography, gamma/beta counters, scintillationcounters and radiographies.

Thus, the sample may be applied to the protein structure for detection,separation and/or quantification of the organic target. This procedureenables not only detection of the organic target, but may in additionshow the distribution and relative level of expression thereof.Optionally, the organic target may be released from the affinity mediumand collected. Thus, the use may comprise affinity purification on anaffinity medium onto which the organic target has been immobilized. Theprotein structure may for example be arranged in a column or in wellplates (such as 96 well plates), or on magnetic beads, agarose beads orsepharose beads. Further, the use may comprise use of the proteinstructures on a soluble matrix, for example using a dextran matrix, oruse in a surface plasmon resonance instrument, such as a Biacore™instrument, wherein the analysis may for example comprise monitoring theaffinity for the immobilized organic target or a number of potentialaffinity ligands.

The protein structures according to the invention can be washed andregenerated with various cleaning agents, including acid, base andchaotropic agents. Particularly useful cleaning agents include NaOH,such as 0.1, 0.5 or 1 M NaOH, and urea, such as 6-8 M urea, Since theprotein structures according to the invention are surprisingly resistantto chemical treatment and/or sterilizing heat treatment, the methodsaccording to the invention involving use of the protein structures maycomprise a final step of regenerating the protein structure. The methodspreferably comprise a final step of regenerating the affinity medium bychemical treatment and/or sterilizing heat treatment. It is preferredthat the chemical treatment comprises treatment with NaOH, such as 0.1,0.5 or 1 M NaOH, and/or urea, such as 6-8 M urea,

Fusion proteins according to the invention can be also be allowed tobind to an organic target in solution, i.e. prior to allowing the fusionprotein to polymerize and form a protein structure, such as a film, afoam or a fibre. Both the spidroin-derived moieties (e.g. REP-CT) assuch and the corresponding fusion proteins incorporating a B moietypolymerise into solid structures even in the presence of contaminatingproteins, without appreciable incorporation of contaminants into thematerial, and the functional (B) moieties retain their expected bindingproperties. It is therefore contemplated that the binding properties ofthe B moiety can be used to capture compounds or cells from thesurrounding solution and incorporate the captured compounds or cellsinto or on a protein structure according to the invention.

Thus, in another preferred method according to the invention, the fusionprotein in the affinity medium is present in solution when contactingthe affinity medium with the sample to achieve binding between theaffinity medium and the organic target. The complex of fusion proteinbound to the organic target is then allowed to form a fusion proteinstructure according to the invention.

This method may be particularly useful when the purpose is to “fish out”specific molecules or cells from a solution, e.g. to obtain targetmolecules from the media in large scale eukaryotic cell productionsystems when the target proteins are secreted. Since the binding oftarget molecules and formation of solid structures by thespidroin-derived moieties can take place at physiological conditions andsince the spidroin-derived moieties are cytocompatible, the method canbe applied repeatedly to an ongoing production process.

The protein structure according to the invention is also useful inseparation, immobilization and/or cultivation of cells. A particularlyuseful protein structure in this respect is a film, a fiber or a foam,see e.g. Example 14 and 23. The film is advantageous in that it adheresto solid structures, e.g. the plastics in microtiter plates. Thisproperty of the film facilitates washing and regeneration procedures andis very useful for separation purposes.

The present invention thus provides a cell scaffold material forcultivation of cells having an organic target that is present on thecell surface. The cell scaffold material is comprising a proteinstructure according to the invention. In certain embodiments, the cellscaffold material is consisting of the protein structure according tothe invention.

It has been found by the present inventors that a cell scaffold materialcomprising a polymer comprising, and optionally consisting of, thefusion protein according to the invention provides a beneficialenvironment for the cultivation of cells, and preferably eukaryoticcells, in a variety of different settings. Furthermore, this environmentenables the establishment of cultures of cells that are otherwise verydifficult, very costly or even impossible to culture in a laboratory,and for the establishment of cell-containing materials useful for tissueengineering and/or transplantation.

The invention also provides a combination of cells, preferablyeukaryotic cells, and the cell scaffold material according to theinvention. Such a combination according to the invention may bepresented in a variety of different formats, and tailored to suit theneeds of a specific situation. It is contemplated, for example, that theinventive combination may be useful as a cell-containing implant for thereplacement of cells in damaged or diseased tissue.

The cell scaffold material can be utilized to capture cells eitherdirectly or indirectly. In direct capture, the B moiety is capable ofselective interaction with an organic target that is present on the cellsurface. Alternatively, the B moiety is capable of selective interactionwith and is bound to an intermediate organic target, and thatintermediate organic target is capable of selective interaction with anorganic target that is present on the cell surface. Thus, in indirectcapture, the cell scaffold material is further comprising anintermediate organic target, and the B moiety is capable of selectiveinteraction with and is bound to said intermediate organic target. Theintermediate organic target, in turn, is capable of selectiveinteraction with the organic target that is present on the cell surface.

In one embodiment of the cell scaffold materials as disclosed herein,the fusion protein is further comprises an oligopeptide cell-bindingmotif. In connection with the cultivation of certain cells in certainsituations, the presence of oligopeptide cell-binding motifs has beenobserved to improve or maintain cell viability, and the inclusion ofsuch a motif into the cell scaffold material as a part of the spidersilk protein is thought to provide additional benefits. The cell-bindingmotif is an oligopeptide coupled to the rest of the fusion protein viaat least one peptide bond. For example, it may be coupled to theN-terminal or the C-terminal of the rest of the fusion protein, or atany position within the amino acid sequence of the rest of the spidersilk protein. With regard to the selection of oligopeptidic cell-bindingmotifs, the skilled person is aware of several alternatives. Saidoligopeptide may for example comprise an amino acid sequence selectedfrom the group consisting of RGD, IKVAV, YIGSR, EPDIM and NKDIL. RGD,IKVAV and YIGSR are general cell-binding motifs, whereas EPDIM and NKDILare known as keratinocyte-specific motifs that may be particularlyuseful in the context of cultivation of keratinocytes. Other usefulcell-binding motifs include GRKRK from tropoelastin, KYGAASIKVAVSADR(laminin derived), NGEPRGDTYRAY (from bone sialoprotein), PQVTRGDVFTMP(from vitronectin), and AVTGRGDSPASS (from fibronectin). The coupling ofan oligopeptide cell-binding motif to the rest of the spider silkprotein is readily accomplished by the skilled person using standardgenetic engineering or chemical coupling techniques. Thus, in someembodiments, the cell-binding motif is introduced via geneticengineering, i.e. forming part of a genetic fusion between a nucleicacid encoding a fusion protein and the cell-binding motif. As anadditional beneficial characteristic of such embodiments, thecell-binding motif will be present in a 1:1 ratio to the monomers offusion protein in the polymer making up the cell scaffold material.

The polymer in the cell scaffold material used in the methods orcombination described herein may adopt a variety of physical forms, anduse of a specific physical form may offer additional advantages indifferent specific situations. For example, in an embodiment of themethods or combination, said cell scaffold material is in a physicalform selected from the group consisting of film, foam, fiber andfiber-mesh.

The present invention accordingly provides a method for immobilizationof cells. A sample e.g. a biological sample such as blood, comprisingcells of interest is provided. The biological sample may be an earlierobtained sample. If using an earlier obtained sample in a method, nosteps of the method are practiced on the human or animal body.

The sample is applied to a cell scaffold material according to theinvention under suitable conditions to allow selective interactionbetween the cell scaffold material and an organic target that is presenton the surface of the cells of interest. The cells are allowed toimmobilize to said cell scaffold material by binding between the organictarget on the cell surface and said cell scaffold material. Non-boundsample is removed under suitable conditions to maintain selectivebinding between the cell scaffold material and the organic target. Thismethod results in cells exhibiting the organic target being immobilizedto the cell scaffold material, and specifically to the proteinstructure, according to the invention.

As set out above, the cell scaffold material can be utilized to capturecells either directly or indirectly. In direct capture, the B moiety iscapable of selective interaction with an organic target that is presenton the cell surface. Alternatively, the B moiety is capable of selectiveinteraction with and is bound to an intermediate organic target, andthat intermediate organic target is capable of selective interactionwith an organic target that is present on the cell surface. Thus, inindirect capture, the cell scaffold material is further comprising anintermediate organic target, and the B moiety is capable of selectiveinteraction with and is bound to said intermediate organic target. Theintermediate organic target, in turn, is capable of selectiveinteraction with the organic target that is present on the cell surface.

Regardless of capture method, the captured cells may be released fromthe fusion protein by cleavage of the fusion protein to release themoiety involved in cell capture from the cell scaffold material. Asmentioned hereinabove, the fusion protein may include a cleavage site inits amino acid sequence, which allows for cleavage and removal of therelevant moiety, typically the B moiety or a cell-binding motif. Variouscleavage sites are known to the person skilled in the art, e.g. cleavagesites for chemical agents, such as CNBr after Met residues andhydroxylamine between Asn-Gly residues, cleavage sites for proteases,such as thrombin or protease 3C, and self-splicing sequences, such asintein self-splicing sequences.

The present invention also provides a method for cultivation of cells.Cells of interest are immobilized to the cell scaffold material usingthe method disclosed hereinabove. The combination of the cell scaffoldmaterial and the immobilized cells are maintained under conditionssuitable for cell culture.

In the context of the present invention, the terms “cultivation” ofcells, “cell culture” etc are to be interpreted broadly, such that theyencompass for example situations in which cells divide and/orproliferate, situations in which cells are maintained in adifferentiated state with retention of at least one functionalcharacteristic exhibited by the cell type when present in its naturalenvironment, and situations in which stem cells are maintained in anundifferentiated state.

The present invention will in the following be further illustrated bythe following non-limiting examples.

EXAMPLES Example 1 Cloning, Expression and Fiber Formation of anIgG-Binding Fusion Protein

To prove the fusion protein concept, a Rep₄CT protein (a REP moiety with4 internal repeats and a CT moiety) was produced in fusion with the Zprotein domain (a B moiety). The Z domain is an engineered version ofthe immunoglobulin G (IgG) binding domain B of staphylococcal protein A,and is a 58 amino acid long triple-helix motif that binds the fragmentcrystallisable (F_(c)) region of IgG. Our aim was to investigate whetherit is possible to produce structures, such as fibers, films andmembranes, from a fusion protein consisting of the Z domain fused toRep₄CT (denoted His₆ZQGRep₄CT, SEQ ID NO: 14) and still retain theIgG-binding ability of domain Z, as well as the structure formingproperties of Rep₄CT. In order to do so a fusion protein consisting ofthe Z domain N-terminally to Rep₄CT was cloned.

Cloning

A gene encoding the His₆ZQGRep₄CT fusion protein (SEQ ID NOS: 14-15) wasconstructed as follows. Primers were designed in order to generate PCRfragments of domain Z from a vector containing such a Z sequence. Also,the primers contained a recognition site for Protease 3C cleavage(LEALFQGP, denoted QG) between Z and Rep₄CT. The resulting PCR productswere then treated with the restriction endonucleases NdeI and EcoRI, aswas the target vector (denoted pT7His₆TrxHis₆QGRep₄CT, harbouring akanamycin resistance gene). Upon restriction cleavage of the targetvector, the TrxHis₆QG part was cleaved off. Cleaved PCR fragments andtarget vector were joined together with the aid of a T4 DNA Ligase,whereupon the resulting, correctly ligated vector (pT7His₆ZQGRep₄CT) wastransformed into chemocompetent Escherichia coli (E. coli) BL21 (DE3)cells that were allowed to grow onto agar plates supplemented withkanamycin (70 μg/ml). Colonies were thereafter picked and PCR screenedfor correct insert and subsequently also sequenced to confirm the DNAsequence inserted of ZQG into the target vector.

Production

E. coli BL21 (DE3) cells possessing the pT7His₆ZQGRep₄CT vector weregrown in Luria-Bertani medium (6 liter in total) supplemented withkanamycin (70 μg/ml) to an OD₆₀₀ value of 1-1.5 in 30° C., followed byinduction of His₆ZQGRep₄CT expression with 300 μM IPTG (isopropylβ-D-1-thiogalactopyranoside) and further incubation in 20° C. forapproximately 2 h. Next, the cells were harvested by a 20 mincentrifugation at 4 700 rpm, and the resulting cell pellets weredissolved in 20 mM Tris (pH 8.0).

Purification

Cell pellets dissolved in 20 mM Tris (pH 8.0) were supplemented withlysozyme and DNase I in order to lyse the bacterial cells, whereupon thecell lysates were recovered after 15 000 rpm of centrifugation for 30min. Next, the recovered cell lysates were divided and loaded onto atotal of four Chelating Sepharose Fast Flow Zn²⁺ columns, keeping theHis₆ZQGRep₄CT protein bound to the column matrix via the His₆ tag. Afterwashing, bound proteins were eluted with 20 mM Tris/300 mM imidazole (pH8.0). The pooled eluate fractions contained 27 mg of His₆ZQGRep₄CTprotein according to an A₂₈₀ measurement. Next, the pooled eluate liquidwas divided into two equal halves (13.5 mg of His₆ZQGRep₄CT in each),where the first half was dialysed against 5 liters of 20 mM Tris (pH8.0) over night, concentrated to 1.07 mg/ml and finally allowed to formfibers. FIG. 3 shows a macroscopic His₆ZQGRep₄CT fiber. The formation offibers from this fusion protein (SEQ ID NO: 14) illustrates that the Zdomain (B moiety) does not interfere with the fiber forming propertiesof Rep₄CT (REP and CT moieties). The amount of His₆ZQGRep₄CT proteinprior to cleavage with Protease 3C was 10 mg after dialysis andconcentration (FIG. 4).

The second half of the eluate pool was cleaved with 1.34 mg of Protease3C, supplemented with 1 mM dithiothreitol (DTT), separating His₆Z fromRep₄CT. The cleavage was performed over night under dialysis against 20mM Tris (pH 8.0), after which the protein solution was allowed to pass aNi-NTA Agarose column, and the flow through fraction, containing Rep₄CT,was collected, concentrated to 0.79 mg/ml and allowed to form fibers.The final amount of Rep₄CT after cleavage was 6 mg (FIG. 4).

FIG. 4 shows an SDS-PAGE gel from the purification of the fusion proteinHis₆ZQGRep₄CT (SEQ ID NO: 14) and its subsequent Protease 3C cleavageproduct Rep₄CT (residues 81-339 of SEQ ID NO: 14). The gel was loaded inthe following order:

(1) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(2) Cell lysate

(3) Flow through from cell lysate loaded onto a Chelating Sepharose FastFlow Zn²⁺ column

(4) Eluate pool of His₆ZQGRep₄CT from a Chelating Sepharose Fast FlowZn²⁺ column

(5) His₆ZQGRep₄CT cleaved with Protease 3C

(6) Flow through of cleaved His₆ZQGRep₄CT loaded onto a Ni-NTA Agarosecolumn

(7) Regeneration of the Ni-NTA Agarose column with 5 ml of 20 mMTris/500 mM imidazole (pH 8.0).

The molecular weights of His₆ZQGRep₃CT, His₆Z, Rep₄CT and Protease 3Care 32 kDa, 9 kDa, 23 kDa and 30 kDa, respectively.

The fact that macroscopic fibers of His₆ZQGRep₄CT (SEQ ID NO: 14) couldbe obtained although Rep₄CT has been fused to another protein, i.e. the58 amino acid long Z domain with binding affinity for IgG, demonstratesthat Rep₄CT still retains its fiber forming properties despite fused tothe Z domain (residues 13-70 of SEQ ID NO: 14). Moreover, the Z domainof the fusion protein seems to confer good solubility to His₆ZQGRep₄CT.

Example 2 Binding of Biotinylated IgG to Fusion Protein Fibers

To further prove the fusion protein concept, it was studied whether theB moiety in a fusion protein structure retains its capacity of selectiveinteraction with an organic target. In this study, the ability of the Zdomain (B moiety) in fibers of the fusion protein His₆ZQGRep₄CT (SEQ IDNO: 14) to bind IgG was assessed. A solution of biotinylated rabbit IgGwas incubated with His₆ZQGRep₄CT fibers, after which the same fiberswere incubated in a solution with streptavidin-functionalized beads, andthe fibers were subsequently visualized in a light microscope. Thechoice of using IgG made in rabbit falls back on the fact that IgG fromrabbit bind with strong affinity to the Z domain.

An approximately 50 mm long His₆ZQGRep₄CT fiber, prepared as describedin Example 1, was immersed in a binding solution containing 50 μl of1×PBS/0.5% bovine serum albumin and 10 μl of 0.5 mg/ml biotinylated IgGproduced in rabbit (anti-rat IgG (H+L), mouse adsorbed, VectorLaboratories, Inc.), and incubated for 75 min in room temperature withlight shaking. The supernatant was discarded, and the fiber was washedthree times in 60 μl 1×PBS/0.07% Tween 20. Next, the fiber was immersedin a solution containing 40 μl of 1×PBS/0.5% bovine serum albumin and 20μl of 10 mg/ml Dynabeads M-280 Streptavidin (Dynal AS), and againincubated for 75 min in room temperature with light shaking. Thesupernatant was discarded and the fiber washed three times in 60 μl1×PBS/0.07% Tween 20.

To get an indication of nonspecific binding of Dynabeads to the fiber,another His₆ZQGRep₄CT fiber was immersed in only a Dynabeads solution,as described above, without the preceding incubation with biotinylatedIgG. The same procedure as described above for His₆ZQGRep₄CT wasperformed with fibers of Rep₄CT type, and all fibers were visualized ina USB microscope with a fixed 500× magnification.

FIG. 5 is a visualization of His₆ZQGRep₄CT and Rep₄CT fibers afterbinding of biotinylated rabbit IgG, followed bystreptavidin-functionalised Dynabeads. Panels (A, B) show tworepresentative pictures of the His₆ZQGRep₄CT fiber, taken at differentpositions along the fiber, that first was incubated with biotinylatedIgG (produced in rabbit), followed by incubation with Dynabeads M-280Streptavidin (Ø 2.8 μm). Panels (C, D) show two representative picturesof another His₆ZQGRep₄CT fiber that was only immersed in a DynabeadsM-280 Streptavidin solution, without the preceding incubation with IgG.The corresponding pictures of Rep₄CT fibers are shown in panels (E, F)and (G, H), respectively. All pictures were taken at a fixed 500×magnification with a USB microscope and the Dynabeads appears in thepictures as dark grey dots.

In FIG. 5, it seems like the Dynabeads are almost only seen in panels Aand B, both these pictures showing a His₆ZQGRep₄CT fiber subjected tobiotinylated IgG, followed by streptavidin-functionalised Dynabeads.This result implies that a considerable fraction of the Z domains in thefusion protein have retained their IgG-binding ability in His₆ZQGRep₄CTafter fiber formation.

Example 3 Binding of Pure and Serum IgG to Fusion Protein Fibers andFilms

To further explore the capacity of the B moiety in a fusion proteinstructure of selective interaction with an organic target, the abilityof domain Z in the fusion protein His₆ZQGRep₄CT (SEQ ID NO: 14) to bindIgG was studied. Fibers and films of this fusion protein were used forbinding of purified IgG and IgG from serum, followed by elution andsubsequent analysis on SDS-PAGE, where IgG under non-reducing conditionsappears as a ˜146 kDa band. Serum is the remaining fluid phase afterblood clotting, and the two main constituents of serum are albumin andIgG. In rabbit serum, for example, the concentration of IgG is 5-10mg/ml and that of albumin even higher. Purified IgG and serum were bothof rabbit source.

Films of His6ZQGRep₄CT were prepared by air-drying 100 μl of proteinsolution (0.96 mg/ml) over night at room temperature at the bottom ofindividual wells of a 24-well tissue culture plate. The casted filmswere then stored at +4° C. for 18 days, either immersed in 20 mM Tris(pH 8.0), denoted “T films”, or without immersed in any liquid, denoted“A films”. FIG. 6 shows part of a casted His₆ZQGRep₄CT film made at thebottom of a well from a hydrophilic, 24-well tissue culture plate. Tocapture the picture, an inverted light microscope at 2× magnificationwas used. Fibers of the fusion protein were prepared as described inExample 1 and stored at +4° C. in 20 mM Tris (pH 8.0) until used.

Two parallel experiment setups were conducted. In the first setup,triplicates of T films, A films and fibers made of His₆ZQGRep₄CT wereimmersed in 500 μl of 50 μg/ml purified rabbit IgG (purified from pooledrabbit sera, Vector Laboratories, Inc.) for 1 h at room temperature withmild shaking. In the other setup, triplicates of the same type ofHis6ZQGRep₄CT films and fibers were instead immersed in 500 μl of a fivetimes dilution of heat-inactivated, centrifuged rabbit serum (NationalVeterinary Institute, Uppsala, Sweden), also for 1 h at room temperaturewith mild shaking. The supernatant was discarded from all fibers andfilms, followed by washing three times in 500 μl 20 mM Tris (pH 8.0).Bound IgG, from purified IgG or from serum, was eluted by 30 min ofincubation in 500 μl of 0.5 M acetic acid/1 M urea/100 mM NaCl (pH 2.7).The same procedure as described above for His₆ZQGRep₄CT fibers and Afilms was also conducted for films and fibers of His₆TrxHis₆QGRep₄CT andRep₄CT as controls. Eluted fractions were analysed with SDS-PAGE undernon-reducing conditions (FIG. 7-9).

FIG. 7 shows a non-reducing SDS-PAGE gel. Eluted fractions were loadedin lanes as follows:

(1-3) His₆TrxHis₆QGRep₄CT, A film, incubated with rabbit IgG

(4-6) His₆TrxHis₆QGRep₄CT, A film, incubated with rabbit serum

(7-8) His₆TrxHis₆QGRep₄CT, fiber, incubated with rabbit IgG

(9) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(10-11) His₆TrxHis₆QGRep₄CT, fiber, incubated with rabbit serum

(12-14) His₆ZQGRep₄CT, T film, incubated with rabbit IgG

(15-17) His₆ZQGRep₄CT, T film, incubated with rabbit serum.

FIG. 8 shows another non-reducing SDS-PAGE gel. Eluted fractions wereloaded according to:

(1-3) His₆ZQGRep₄CT, A film, incubated with rabbit IgG

(4-6) His₆ZQGRep₄CT, A film, incubated with rabbit serum

(7-9) His₆ZQGRep₄CT, fiber, incubated with rabbit IgG

(10) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(11-13) His₆ZQGRep₄CT, fiber, incubated with rabbit serum

(14-16) Rep₄CT, A film, incubated with rabbit IgG.

FIG. 9 shows a further non-reducing SDS-PAGE gel. Eluted fractions wereloaded according to:

(1-3) Rep₄CT, A film, incubated with rabbit serum

(4-6) Rep₄CT, fiber, incubated with rabbit IgG

(7) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(8-10) Rep₄CT, fiber, incubated with rabbit serum

(11) Purified rabbit IgG (50 μg/ml), used in incubation

(12) Rabbit serum (1:50 dilution), used in incubation.

The results in FIG. 7-9 show that all types of His₆ZQGRep₄CT matrices,i.e. films (both A and T type) and fibers have the ability to bind IgG(either purified or from serum) via the Z domain. Moreover, theHis₆ZQGRep₄CT matrices exposed to rabbit serum do not seem to bindanything else of the serum fraction but IgG. Matrices of the other twoprotein variants used (i.e. His₆TrxHis₆QGRep₄CT and Rep₄CT) do not seemto bind anything at all, except from fibers of Rep₄CT exposed to serum,and that show weak bands within the IgG (˜146 kDa) and albumin (˜70 kDa)region. This approach to evaluate the IgG-binding ability of the Zdomain within the fusion protein His₆ZQGRep₄CT (SEQ ID NO: 14) is astrong indication that the Z domain is active in both fiber and filmversions of the fusion protein. No other fraction of rabbit serum thanIgG is observed to bind to His₆ZQGRep₄CT.

Example 4 Binding Reproducibility of Pure and Serum IgG to FusionProtein Fibers and Films

To explore the reproducibility of the IgG binding to films and fibers ofthe fusion protein His₆ZQGRep₄CT (SEQ ID NO: 14), the experiments inExample 3 were performed again. The same fibers and films ofHis₆ZQGRep₄CT, His₆TrxHis₆QGRep₄CT and Rep₄CT that were used in Example3 were used again. All fiber and film materials had been immersed for 70days in 20 mM Tris (pH 8.0) at +4° C. after the previous experiments hadbeen performed. The experiments were carried out as described in Example3. Eluted fractions were analysed with SDS-PAGE under non-reducingconditions (FIGS. 10-12).

FIG. 10 shows a non-reducing SDS-PAGE gel. Eluted fractions were loadedaccording to:

(1-3) His₆TrxHis₆QGRep₄CT, A film, incubated with rabbit IgG

(4-6) His₆TrxHis₆QGRep₄CT, A film, incubated with rabbit serum

(7-8) His₆TrxHis₆QGRep₄CT, fiber, incubated with rabbit IgG

(9) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(10-11) His₆TrxHis₆QGRep₄CT, fiber, incubated with rabbit serum

(12-14) His₆ZQGRep₄CT, T film, incubated with rabbit IgG

(15-17) His₆ZQGRep₄CT, T film, incubated with rabbit serum.

FIG. 11 shows another non-reducing SDS-PAGE gel. Eluted fractions wereloaded according to:

(1-3) His₆ZQGRep₄CT, A film, incubated with rabbit IgG

(4-6) His₆ZQGRep₄CT, A film, incubated with rabbit serum

(7-9) His₆ZQGRep₄CT, fiber, incubated with rabbit IgG

(10) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(11-13) His₆ZQGRep₄CT, fiber, incubated with rabbit serum

(14-16) Rep₄CT, A film, incubated with rabbit IgG.

FIG. 12 shows a non-reducing SDS-PAGE gel. Eluted fractions were loadedaccording to:

(1-3) Rep₄CT, type A film, incubated with rabbit serum

(4-6) Rep₄CT, fiber, incubated with rabbit IgG

(7) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(8-10) Rep₄CT, fiber, incubated with rabbit serum

(11) Empty well

(12) Purified rabbit IgG (50 μg/ml), used in incubation

(13) Rabbit serum (1:50 dilution), used in incubation.

The IgG binding patterns for all three protein matrices (i.e.His₆ZQGRep₄CT, His₆TrxHis₆QGRep₄CT and Rep₄CT) corresponds to what wasobserved in Example 3, although rather weak bands corresponding toalbumin (˜70 kDa) is seen for His₆ZQGRep₄CT fibers incubated with rabbitserum. This initial IgG-binding reproducibility study, show that bothfibers and films of His₆ZQGRep₄CT can be used at least twice for bindingand elution of purified and serum IgG. A further study ofreproducibility is reported in Example 19.

Example 5 IgG Binding to Fusion Protein Matrices and to a CommercialProtein a Matrix

The IgG-binding capacity of His₆ZQGRep₄CT fusion protein structures wasevaluated by comparison with a commercially available protein A matrix(Protein A Sepharose CL-4B, GE Healthcare). Fibers and films ofHis₆ZQGRep₄CT (SEQ ID NO: 14) were prepared inside spin columns. Theprotein A matrix was also added to spin columns in such a way that thetotal number of protein A molecules attached to the matrix was equal tothe total number of Z molecules within His₆ZQGRep₄CT films. Binding ofpurified IgG and IgG from serum to films and fibers of His₆ZQGRep₄CT, aswell as to the protein A matrix was allowed to occur, followed byelution and subsequent analysis on SDS-PAGE.

Films of His₆ZQGRep₄CT were prepared by air-drying 100 μl of proteinsolution (1.05 mg/ml) for three days at room temperature at the bottomof the polyethylene frit inside spin columns (SigmaPrep™ Spin Columns,Sigma), giving a total of 3×10⁻⁹ mole of His₆ZQGRep₄CT per film. Also,fibers of His₆ZQGRep₄CT were placed onto frits inside the same type ofspin columns.

The same procedure was carried out for films and fibers of Rep₄CT, wherethe films contained a total of 4×10⁻⁹ mole of Rep₄CT per film. For thecommercial protein A matrix, a drained matrix volume corresponding to3×10⁻⁹ mole of protein A was transferred to the frit per spin column,whereupon the matrix was washed with 1×500 μl plus 2×150 μl of deionizedwater by centrifugation of the spin columns at 400 rcf for 1.5 min.

Two parallel experiment setups were conducted for fibers and films ofHis₆ZQGRep₄CT and Rep₄CT, as well as with the protein A matrix. In thefirst setup, duplicates of all three different matrices were immersed in500 μl of 50 μg/ml purified rabbit IgG (IgG purified from rabbit serum,Sigma) for 1 h at room temperature. In the other setup, duplicates ofthe same three types of matrices were instead immersed in 500 μl of afive times dilution of centrifuged rabbit serum (Normal rabbit serum,Invitrogen), also for 1 h at room temperature.

The supernatant was discarded from all fibers and films by simplepipetting and for the protein A matrix by centrifugation (400 rcf, 1.5min), followed by washing three times in 500 μl 20 mM Tris (pH 8.0).Bound IgG, from purified IgG or from serum, was eluted by 30 min ofincubation in 500 μl of 0.5 M acetic acid/1 M urea/100 mM NaCl (pH 2.7).Eluted fractions were analysed on SDS-PAGE under non-reducing conditions(FIGS. 13-15), and are denoted as coming from run 1. Immediately afterthe elution, all matrices were washed with 3×500 μl 20 mM Tris (pH 8.0),after which the just described experiment was repeated once more toevaluate the reproducibility of IgG binding. Eluted fractions from therepeated experiment are denoted as coming from run 2. Note: Undernon-reducing SDS-PAGE conditions, the molecular weight of IgG is around146 kDa.

FIG. 13 shows a non-reducing SDS-PAGE gel of eluted fractions from run1, loaded according to:

(1) Purified rabbit IgG (50 μg/ml), used in incubation

(2) Rabbit serum (1:50 dilution), used in incubation

(3-4) His₆ZQGRep₄CT, film, incubated with rabbit IgG

(5-6) His₆ZQGRep₄CT, film, incubated with rabbit serum

(7-8) Rep₄CT, film, incubated with rabbit IgG

(9) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(10-11) Rep₄CT, film, incubated with rabbit serum

(12-13) Protein A Sepharose CL-4B matrix, incubated with rabbit IgG

(14-15) Protein A Sepharose CL-4B matrix, incubated with rabbit serum

(16-17) His₆ZQGRep₄CT, fiber, incubated with rabbit IgG.

FIG. 14. shows a non-reducing SDS-PAGE gel of eluted fractions comingfrom both run 1 and run 2, loaded according to:

(1-2) His₆ZQGRep₄CT, fiber, incubated with rabbit serum, run 1

(3-4) Duplicates of Rep₄CT, fiber, incubated with rabbit IgG, run 1

(5-6) Duplicates of Rep₄CT, fiber, incubated with rabbit serum, run 1

(7) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(8) Purified rabbit IgG (50 μg/ml), used in incubation

(9) Rabbit serum (1:50 dilution), used in incubation

(10-11) His₆ZQGRep₄CT, film, incubated with rabbit IgG, run 2

(12-13) His₆ZQGRep₄CT, film, incubated with rabbit serum, run 2

(14-15) Rep₄CT, film, incubated with rabbit IgG, run 2

(16-17) Rep₄CT, film, incubated with rabbit serum, run 2.

FIG. 15 shows a non-reducing SDS-PAGE gel of eluted fractions, allcoming from run 2 and loaded according to:

(1-2) Protein A Sepharose CL-4B matrix, incubated with rabbit IgG

(3-4) Protein A Sepharose CL-4B matrix, incubated with rabbit serum

(5-6) His₆ZQGRep₄CT, fiber, incubated with rabbit IgG

(7) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(8-9) His₆ZQGRep₄CT, fiber, incubated with rabbit serum

(10-11) Rep₄CT, fiber, incubated with rabbit IgG

(12-13) Rep₄CT, fiber, incubated with rabbit serum.

The results in FIG. 13-15 show that the matrices selectively bind IgGfrom serum. The IgG-binding capacity of all types of His₆ZQGRep₄CTmatrices, i.e. films and fibers, is in the same range as the commercialprotein A matrix. Similarly to the commercial protein A matrix, thefusion protein structures can be regenerated with maintained bindingcapacity.

Example 6 Cleaning-in-Place (CIP) of Fusion Protein Matrices and aCommercial Protein a Matrix

To evaluate whether precipitated or denatured substances remain attachedto protein structures made of His₆ZQGRep₄CT (SEQ ID NO: 14) and Rep₄CTand to a commercial protein A matrix (Protein A Sepharose CL-4B, GEHealthcare) after elution, a cleaning-in-place (CIP) with 8 M urea wascarried out for all the matrices exposed to rabbit serum used in theexperiments in Examples 4-5.

Fibers and films made of His₆ZQGRep₄CT and Rep₄CT from Example 4 andExample 5, and a commercial Protein A matrix from Example 5, allpreviously exposed to rabbit serum, were immersed in 200 μl of 8 M ureaat room temperature for 20 min, prior to supernatant removal, andsubsequent analysis of urea fractions on SDS-PAGE under non-reducingconditions (FIG. 16-17).

FIG. 16 shows a non-reducing SDS-PAGE gel from cleaning-in-place with 8M urea of matrices subjected twice to rabbit serum. The gel was loadedaccording to:

(1) Rabbit serum (1:50 dilution)

(2) Empty well

(3-5) His₆ZQGRep₄CT, T film, from Example 4

(6-8) His₆ZQGRep₄CT, A film, from Example 4

(9) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(10-12) His₆ZQGRep₄CT, fiber, from Example 4

(13-15) Rep₄CT, A film, from Example 4

(16) Rep₄CT, fiber, from Example 4

(17) Empty well.

FIG. 17 shows a second non-reducing SDS-PAGE gel from cleaning-in-placewith 8 M urea of matrices subjected twice to rabbit serum. The gel wasloaded according to:

(1) Rabbit serum (1:50 dilution)

(2) Empty well

(3-4) Rep₄CT, fiber, from Example 4

(5-6) His₆ZQGRep₄CT, film, from Example 5

(7) Rep₄CT, film, from Example 5

(8) Spectra Multicolor Broad Range Protein Ladder, Fermentas

(9) Rep₄CT, film, from Example 5

(10-11) His₆ZQGRep₄CT, fiber, from Example 5

(12-13) Rep₄CT, fiber, from Example 5

(14-15) Protein A Sepharose CL-4B matrix, from Example 5

(16-17) Empty wells.

The results in FIG. 16-17 indicate that only low amounts of precipitatedor denatured substances remain attached to the fusion protein structuresafter elution and cleaning. In particular, only low amounts ofprecipitated or denatured substances, in the same range as for thecommercial protein A matrix, remain attached to films of theHis₆ZQGRep₄CT fusion protein.

Example 7 Cloning, Expression and Fiber Formation of an Albumin-BindingFusion Protein

To further prove the fusion protein concept, Rep₄CT was produced infusion with the albumin binding domain (Abd) from streptococcal proteinG. Abd is a 5-kDa triple-helix motif that binds albumin. In order to doso a fusion protein consisting of the Abd domain N-terminally to Rep₄CT(denoted His₆AbdQGRep₄CT) was cloned (SEQ ID NOS: 16-17).

Cloning

Primers were designed in order to generate PCR fragments of Abd from avector containing such a sequence. Also, the primers contained aProtease 3C cleavage site, denoted QG, between Abd and Rep₄CT. Theresulting PCR products were then treated with the restrictionendonucleases NdeI and EcoRI, as was the target vector, denotedpT7His₆TrxHis₆QGRep₄CT (harbouring a kanamycin resistance gene). Uponrestriction cleavage of the target vector, the TrxHis₆QG part wascleaved off. Cleaved PCR fragments and target vector were joinedtogether with the aid of a T4 DNA Ligase, whereupon the resulting,correctly ligated vector (pT7His₆AbdQGRep₄CT) was transformed intochemocompetent E. coli BL21 (DE3) cells that were allowed to grow ontoagar plates supplemented with kanamycin. Colonies were thereafter pickedand PCR screened for correct insert and subsequently also sequenced.

Production

E. coli BL21 (DE3) cells possessing the pT7His₆AbdQGRep₄CT vector weregrown in Luria-Bertani medium (6 liters in total) supplemented withkanamycin to an OD₆₀₀ of 1-1.5 in 30° C., followed by induction ofHis₆AbdQGRep₄CT expression with IPTG and further incubation in 20° C.for approximately 2 h. Next, the cells were harvested by centrifugation,and the resulting cell pellet was dissolved in 20 mM Tris (pH 8.0).

Purification

Cell pellets dissolved in 20 mM Tris (pH 8.0) were supplemented withlysozyme and DNase I in order to completely lyse the bacterial cells,whereupon the supernatants were recovered after 15 000 rpm ofcentrifugation. Next, the recovered supernatants were loaded onto a NiIMAC column or a Chelating Sepharose Fast Flow ZN column, keeping theHis₆AbdQGRep₄CT protein bound to the matrix via the His₆ tag. Afterwashing, bound proteins were eluted with 20 mM Tris/300 mM imidazole (pH8.0). The pooled eluate fractions, containing His₆AbdQGRep₄CT (SEQ IDNO: 16) was dialyzed against 5 liters of 20 mM Tris (pH 8.0),concentrated, and a final amount of 4 mg protein was obtained.

Fiber and Film Formation

From the purified, soluble Abd-Rep₄CT protein, both fibers and filmswere successfully made at a protein concentration of 0.87 mg/ml

The fact that macroscopic fibers and films of Abd-Rep₄CT were obtainedalthough Rep₄CT had been fused to another protein, namely the albuminbinding domain (Abd), demonstrates that Rep₄CT retains its fiber formingproperties despite fused to the Abd domain. Next, the aim was to revealwhether the Abd domain had retained its albumin-binding ability whenfused to Rep₄CT, see Examples 24 and 25.

Example 8 Cloning, Expression and Fiber Formation of an IgG-BindingFusion Protein

To further prove the fusion protein concept, Rep₄CT was produced infusion with the IgG binding domain C2 from streptococcal protein G. C2contains 55 amino acids, and the structure is constituted of twoβ-hairpins that are associated to form a four stranded mixedantiparallel/parallel β-sheet with a single α-helix lying across oneface of the sheet. In order to do so a fusion protein consisting of theC2 domain N-terminally to Rep₄CT (denoted His₆C2QGRep₄CT) was cloned(SEQ ID NOS: 18-19).

Cloning

Primers were designed in order to generate PCR fragments of C2 from avector containing such a sequence. Also, the primers contained aProtease 3C cleavage site, denoted QG, between C2 and Rep₄CT. Theresulting PCR products were then treated with the restrictionendonucleases NdeI and EcoRI, as was the target vector, denotedpT7His₆TrxHis₆QGRep₄CT (harbouring a kanamycin resistance gene). Uponrestriction cleavage of the target vector, the TrxHis₆QG part wascleaved off. Cleaved PCR fragments and target vector were joinedtogether with the aid of a T4 DNA Ligase, whereupon the resulting,correctly ligated vector (pT7His₆C2QGRep₄CT) was transformed intochemocompetent E. coli BL21 (DE3) cells that were allowed to grow ontoagar plates supplemented with kanamycin. Colonies were thereafter pickedand PCR screened for correct insert and subsequently also sequenced.

Production

E. coli BL21 (DE3) cells possessing the pT7His₆C2QGRep₄CT vector weregrown in Luria-Bertani medium (6 liters in total) supplemented withkanamycin to an OD600 of 1-1.5 in 30° C., followed by induction ofHis₆C2QGRep₄CT expression with IPTG and further incubation in 20° C. forapproximately 2 h. Next, the cells were harvested by centrifugation andthe resulting cell pellet dissolved in 20 mM Tris (pH 8.0).

Purification

Cell pellets dissolved in 20 mM Tris (pH 8.0) were supplemented withlysozyme and DNase I in order to completely lyse the bacterial cells,whereupon the supernatants were recovered after 15 000 rpm ofcentrifugation. Next, the recovered supernatants were loaded onto a NiIMAC column, keeping the His₆C2QGRep₄CT protein bound to the matrix viathe His₆ tag. After washing, bound proteins were eluted with 20 mMTris/300 mM imidazole (pH 8.0). The pooled eluate fractions, containingHis₆C2QGRep₄CT was dialyzed against 5 liters of 20 mM Tris (pH 8.0),concentrated, and a final amount of 6 mg protein was obtained.

Fiber and Film Formation

From the purified, soluble C2-Rep₄CT protein, both fibers and films weresuccessfully made at a protein concentration of 0.87 mg/ml. The factthat macroscopic fibers and films of C2-Rep₄CT was obtained althoughRep₄CT had been fused to another protein, namely the IgG binding domainC2, demonstrates that Rep₄CT retains its fiber forming propertiesdespite being fused to the C2 domain. Next, the aim was to revealwhether the C2 domain had retained its IgG-binding ability when fused toRep₄CT, see Examples 26 and 27.

Example 9 Cloning, Expression and Formation of Films and Fibers of aBiotin-Binding Fusion Protein

Streptavidin is a tetramer of four identical monomers with one bindingsite per monomer. It shows high affinity towards biotin (vitamin H),reaching a dissociation constant of K_(d)˜10⁻¹⁵ M, making this anessentially irreversible binding event. Streptavidin also shows a highstability in presence of proteases, at elevated temperatures and indenaturing agents, and at extreme pH values (Wilchek, M. et al., Anal.Biochem. 171: 1-32 (1988)). Thus, this interaction is attractive in manyapplications including protein labeling, separation and targeting. Inpractice, biotinylation is nowadays easily facilitated utilizingbiotinylated linker molecules that also houses one out of severalreactive organic molecules that attacks and bridge the biotin todifferent biological molecules, e.g. proteins and DNA. However theproduction of high density functional surfaces coated with Streptavidinhas proven difficult to achieve, see e.g. Table 4. To reduce the bindingstrengths in applications where reversibility in binding is essential(e.g. during purification) and to be able to successfully expresssoluble protein in E. coli, a monomeric variant of streptavidin has beendeveloped, M4 (Wu. S.-C. et al., Protein Expres. Purif. 46, 268-273(2006)). Compared to the monomers of the wild type tetramerstreptavidin, M4 has four amino acid substitutions (V55T, T76R, L109Tand V125R), which keep M4 in an active monomeric form.

M4 was N- or C-terminally fused to Rep₄CT (SEQ ID NOS: 20-21) byrecombinant techniques. The resulting proteins and genes encoding themwere named M4Rep₄CT (SEQ ID NOS: 22-23), modM4Rep₄CT (SEQ ID NOS: 24-25)and Rep₄CTM4 (SEQ ID NOS: 26-27), respectively. The difference betweenM4Rep₄CT and modM4Rep₄CT is the substitution of a Gly to Arg-Ala-Arg inthe linker region between M4 and Rep₄CT. All proteins were expressedfused to a His₆-Trx-His₆ tag that was cleaved off and removed duringpurification.

Production and purification of all proteins were performed essentiallyas described in Stark, M. et al., Biomacromolecules 8, 1695-1701 (2007)and Hedhammar M. et al., Biochemistry 47, 3407-3417 (2008). The proteinconcentration of Rep₄CTM4, M4Rep₄CT and modM4Rep₄CT was measured at 280nm using a molar extinction coefficient of 53860 M⁻¹ cm⁻¹. The purifiedprotein sample was subjected to reducing SDS-PAGE, and protein puritywas determined after staining of the gel with Coomassie Brilliant BlueR-250. The theoretical molecular weights of all the purified proteinsand other relevant physical properties are listed in Table 3.

TABLE 3 Physical parameters of the expressed proteins Amino MolecularMolar extinction SEQ acids weight* coefficient* Protein ID NO (#)(g/mol) (M⁻¹ cm⁻¹) Rep₄CT 20 263 23053 11920 Rep₄CTM4 26 428 40157 53860M4Rep₄CT 22 427 40100 53860 modM4Rep₄CT 24 429 40427 53860 *Theoreticalvalues

From each of the Rep₄CTM4, M4Rep₄CT and modM4Rep₄CT fusion proteins,both film and fibers were formed. These results confirm that Rep₄CTretains its ability to self assemble into solid structures althoughfused to M4. This also confirms that it is possible to obtain fibers andfilms of proteins where M4 is fused to Rep₄CT using linkers of differentlengths.

Example 10 Binding of a Biotin-Containing Target to Fusion ProteinFibers and Films

(A) Binding of Biotinylated Atto-565 to Rep₄CTM4 Films.

Rep₄CTM4 (SEQ ID NO: 26) and Rep₄CT (SEQ ID NO. 20, control) wereallowed to form films by drying 25 μl of protein solution in roomtemperature in the bottom of wells in clear or black 96 well microtiterplates. The plates were stored at room temperature for one to two weeksbefore use. The wells were incubated with 100 μl 1% BSA in PBS (pH 7.4)for >1 hour at room temperature in order to avoid non-specific binding.Background values were obtained by measuring fluorescence intensity inthe wells with films of the respective protein, Rep₄CT or Rep₄CTM4, in50 μl phosphate buffered saline (PBS) prior to addition of biotinylatedAtto-565. The wells were further incubated with 50 μl of an 80 μMsolution of biotinylated Atto-565 (Sigma Aldrich, Germany) dissolved in1% BSA in PBS (pH 7.4). The mixture was allowed to stand in roomtemperature between two to three hours before washing twice with PBScontaining 0.05% Tween-20 (PBS-T) and once with PBS. The resultingfluorescence intensity was recorded after adding 50 μl of PBS into thewells in a Tecan Infinite M200 microplate reader (λ_(ex)=565 nm,λ_(em)=590 nm).

A dilution series was prepared in PBS, and 50 μl of samples of differentconcentrations of biotinylated Atto-565 was added to wells with therespective films, in triplicates, whereafter the resulting fluorescenceintensity from the wells was recorded.

In FIG. 18, the fluorescence intensities from protein films aftersoaking with biotinylated Atto-565 and washing are observed in 2×magnification using a Nicon Eclipse Ti—S fluorescence microscope(λ_(ex)=509-550 nm and λ_(em)=570-614 nm). The difference influorescence intensity between Rep₄CTM4 films (panel A) and Rep₄CT films(panel B) after addition of biotinylated Atto-565 is evident.

In order to establish the total amount of biotinylated Atto-565 bindingto the Rep₄CTM4 films, fluorescence intensity was recorded using adilution series of known amounts of biotinylated Atto-565. In this way,the fluorescence intensities from samples with known amounts (in moles)biotinylated Atto-565 (in triplicates, in wells with Rep₄CTM4) were usedto obtain a standard curve. The resulting fluorescence intensity valuescorresponding to background value (Rep₄CTM4 films without biotinylatedAtto-565) and data points correlating to three concentrations ofbiotinylated Atto-565 added to the wells are shown in the graph in FIG.19. The table below the graph in FIG. 19 show the values obtained from alinear regression to the fluorescence intensity values, with standarddeviations from measurement in n=3 wells with the same biotinylatedAtto-565 concentration.

Starting from the fluorescence intensity values resulting from bindingexperiments of biotinylated Atto-565 to films of Rep₄CTM4 (FIG. 20),these values were used to calculate the amount of moles of biotinylatedAtto-565 that correspond to the obtained fluorescence intensity. Theresulting fluorescence values are plotted in FIG. 20, with panels A andB displaying values before (−) and after (+) addition of biotinylatedAtto-565 to wells with films of Rep₄CT (A) and Rep₄CTM4 (B). Panel Cshows a comparison of the resulting fluorescence intensities afteraddition of biotinylated Atto-565 to films of Rep₄CT or Rep₄CTM4. Therewas no significant (ns) difference between before and after values forthe Rep₄CT films (panel A). Significant differences between before andafter addition of biotinylated Atto-565 values with Rep₄CTM4 (panel B;P<0.01), and between proteins (panel C; P<0.0001), were confirmed bystatistical tests. The bars in FIG. 20 indicate standard deviationbetween fluorescence intensity values of n=10 films.

The biotinylated fluorophore/surface area ratio obtained from thebinding experiments was calculated. To surface areas of approximately 28mm², 2.1 μmol biotinylated Atto-565 was bound to the Rep₄CTM4 films.This results in a bound biotin/surface area of 0.073 μmol/mm² (Table 5).

(B) Binding of Biotinylated Horse Radish Peroxidase (HRP) to Rep₄CTM4Films

Due to its relatively high stability and the production of chromogenicproducts in the conversion of a non-chromogenic substrate and peroxide,HRP is commonly used coupled to a secondary antibody or a bindermolecule (e.g. biotin) in applications such as ELISAs, Western blots andimmunohisto-chemistry. In order to establish the total amount ofbiotinylated HRP binding to films made of Rep₄CTM4 (SEQ ID NO: 26) andRep₄CT (SEQ ID NO: 20; control), biotinylated HRP was allowed to bind toeach respective film. The rate of product formation was recorded at 570nm using known amounts of substrates. The molar extinction coefficientfor the product, resorufin, was obtained from the manufacturer(Invitrogen), stated to be 54000 cm⁻¹M⁻¹.

Protein solutions (25 μl) of Rep₄CTM4 and Rep₄CT were allowed tocompletely dry in the bottom of wells in clear 96 well microtiterplates, thus forming films. The wells were incubated with 100 μl of 1%BSA in PBS (pH 7.4) for >1 hour before incubation with 50 μl of a 0.3mg/ml biotinylated HRP (Invitrogen, Camarillo, Calif.) in 1% BSA in PBS(pH 7.4) for >1 hour. The wells were subsequently washed twice withPBS-T and once with PBS. Reactions were initiated by addition of 50 μlof a 50 μM Amplex red solution (Invitrogen) with 2 mM hydrogen peroxidedissolved in 0.2% BSA, 28 mM NaCl, 0.54 mM KCl, 0.3 mM KH₂PO₄, 42 mMNa₂HPO₄ (pH 7.4) at room temperature. Kinetic measurements wereconducted on a Tecan Infinite M200 microplate reader.

Known amounts of biotinylated HRP (free in solution) were used toestablish which amount of HRP that resulted in the same rate of productformation as in measurements with biotinylated HRP on the films. Theresulting reaction velocities in catalysis by biotinylated HRP free insolution (pH 7.4) of 50 μM Amplex red and 2 mM hydrogen peroxide to theproduct, resorufin, are shown in the graph in FIG. 21. Data pointscorrelates to triplicate measurements with the same concentration ofbiotinylated HRP. The table below the graph in FIG. 21 shows the valueobtained from a linear regression to the data. The bars in the graph inFIG. 21 indicate standard deviations from measurements in n=3 wells withthe same concentration of biotinylated HRP. The resulting standard curveand slope in FIG. 21 was used for calculation of the amount ofbiotinylated HRP that was bound to the fusion protein films.

The reaction velocities in catalysis of 50 μM Amplex red and 2 mM H₂O₂in wells where films of the fusion protein and control were incubatedwith biotinylated HRP is shown in FIG. 22. The reaction velocities inFIGS. 21 and 22 are expressed as the formation of resorufin in μM perminute, calculated using the extinction coefficient provided byInvitrogen (54 000 cm⁻¹M⁻¹). Bars indicate standard deviation forreactions measured in n=8 wells. FIG. 22 shows a significant differencein rate of product formation (and hence also bound biotinylated HRP)between wells coated with Rep₄CTM4 and the controls, Rep₄CT-coatedwells. It was determined that 0.2 μmol HRP/mm² was bound to the Rep₄CTM4films, (Table 5).

(C) Comparison with Commercial Products

The production of high density functional surfaces coated withStreptavidin has proven difficult to achieve. The biotin bindingcapacities of commercially available plates are listed in Table 4.

TABLE 4 Biotin binding capacities of commercially available plates CoatBiotin/coat Biotin^(a) area^(b) area Company Plate (pmol) (mm²)(pmol/mm²) Nunc/Thermo Passively 13 150 0.087 scientific Coated platesPierce/Thermo HBC^(c) 0.78^(d)-125  90 0.008-1.4 scientificPierce/Thermo Standard  5  90 0.055 scientific Capacity Grenier c-bottom20 210 0.096 bio-one R&D systems EvenCoat   7^(e) 150 0.047 Sigmascreen/ S6940 300  150 2.0  Sigma Aldrich (HBCc) ^(a)Biotin-bindingcapacity as stated by manufacturer ^(b)Calculated based on coat volumeas stated by manufacturer ^(c)HBC = high binding capacity. ^(d)Thelimits were calculated based on stated detection range of a 8 kDabiotinylated molecule. ^(e)Calculation based on stated binding ofbiotinylted antibodies using a MW = 120 kDa.

The biotinylated fluorophore/surface area ratio obtained from thebinding experiments with biotinylated Atto-565 in Example 10 A andbiotinylated HRP in Example 10 B are summarized in Table 5. Thedensities of the two different biotinylated molecules on films ofRep₄CTM4 (SEQ ID NO: 26) are 0.073-0.2 μmol/mm².

TABLE 5 Biotin binding capacities of films of M4 fusion protein Bio-Sur- Biotinylated Bio- Biotin- tinylated face molecule/ tinylated ylatedRep₄CTM4 molecule area surface area Rep₄CTM4 product (nmol) (pmol) (mm²)(pmol/mm²) (%) Atto- 0.082 2.1 28 0.073 2.6 565 HRP 0.082 5.1 28 0.2 6.2

The results in Tables 4-5 indicate that films made of fusion proteinswith a M4 moiety provide a biotin binding density in the same ranges asfor commercial alternatives, regardless of whether the biotin is coupledto a small (fluorophore) or large (protein) molecule.

Statistics

GraphPad Prism 4.0 (GraphPad Software, San Diego, Calif.) was used forstatistical analysis of data. In Example 10 A, a non-parametric pairedWilcoxon test was used in the comparison between fluorescence valuesbefore and after addition of biotinylated Atto-565 to films formed fromeither Rep₄CT or Rep₄CTM4. Further, a non-parametric, unpaired MannWhitney U test was used to compare fluorescence intensity in wells afterincubation of biotinylated Atto-565 using Rep₄CT or Rep₄CTM4 film, andalso to compare the [resorufin]/min values obtained from measurements onfilms incubated with biotinylated HRP in Example 10 B. P-values<0.05were considered significant.

Example 11 Binding of a Biotionylated Antibody and a Secondary Antibodyto Fusion Protein Fibers and Films

Film and fibers of Rep₄CT (SEQ ID NO: 20, control) and modM4Rep₄CT (SEQID NO: 24) are tested for binding capacity to a biotinylated antibody ofrabbit origin. The films are formed in 8×1 or 12×1 well strips (wells inequal size to the 96 well microplate formats).

After preincubation of the films and fibers with 1% BSA in PBS (pH 7.4),the biotinylated antibody (rabbit) is incubated with the proteinstructures (film/fiber) for >1 h, followed by addition of a secondaryanti-rabbit antibody, radioactively labeled with ¹²⁵I. The films andfibers are washed. Detection of the gamma radiation is carried out onindividual films or individual fibers in a gamma counter. A dilutionseries of known amounts of ¹²⁵I-labeled antibody is prepared, andradiation is measured to obtain a standard curve from which the amountof bound biotinylated antibody to the fusion protein films and fiberscan be calculated.

Example 12 Preparation of a Pure Film of Fusion Protein

Films were casted using 25 μl of RepCT₄M4 (SEQ ID NO: 26) at aconcentration of 10-20 μM. 100 μl of PBS (pH 7.4) was allowed toincubate in wells with film for one hour. This solution was removed, and50 μl hexafluoroisopropanol (HFIP) was added to break the film andsolubilize the protein. To additional films, the same amount of HFIP wasadded without prior washing with PBS. The HFIP was allowed to work onthe four films for 3.5 h, and the resulting clear HFIP solution wastransferred into Eppendorf tubes containing 50 μl of 20% SDS. The waterin the tubes was evaporated, and the remaining content was dissolved in60 μl 10 mM Tris-HCl (pH 8.0) and subjected to reducing SDS-PAGE.

The results are shown in FIG. 23. Lane 1 corresponds to the proteins inthe Spectra™ Multicolor Broad Range Protein Ladder (Fermentas). Numberscorrespond to protein sizes. Lane 2 corresponds to purified Rep₄CTM4films that were not soaked with PBS before adding the HFIP. Lane 3corresponds to a Rep₄CTM4 film which has been soaked in PBS for one hourbefore removing this solution and thereafter adding HFIP.

Although some proteins are co-purified with the Rep₄CTM4 protein (lane2, FIG. 23), incubation with 100 μl PBS for one hour dissolvescontaminating proteins leaving a film consisting only of Rep₄CTM4proteins, as judged by reducing SDS-PAGE (lane 3, FIG. 23). Thus, mostcontaminating proteins are removed by the polymerization process assuch, and any remaining impurities in proteins structures made of fusionproteins can easily be removed by gentle washing with aqueous buffers.

Example 13 Capture of Organic Targets in Solution

A ZRep₄CT protein solution (˜1 mg/ml) is added to a sample consisting ofserum, whereby the IgG molecules in the sample are allowed to bind tothe Z moiety of the fusion protein in solution. The mixture is subjectedto a hydrophobic/hydrophilic interface which causes the Rep₄CT part ofthe fusion protein/Ig G complex to form a film or a foam, leaving otherserum proteins in solution and capturing the IgG on a solid structure.

Alternatively, the mixture is allowed to dry on a solid support, forminga film with immobilized IgG. This film is washed with a buffer, e.g.PBS, to dissolve and remove contaminating proteins.

Alternatively, fibers are formed by providing a hydrophobic-hydrophilicinterface and subjecting the mixture to shear forces. The Rep₄CT part ofthe fusion protein/IgG complex polymerises into macroscopic fibers,leaving other proteins in the solution.

The solid structures formed (films, foams or fibers) may be collected,and the IgG can be recovered in a suitable elution buffer (e.g. bylowering the pH to 2.7). Eluted proteins are identified on SDS-PAGE. Seealso Example 22.

Example 14 Fusion Protein Scaffolds for Cell Capture

(A) In Vivo Studies of Non Adherent Cells in the Anterior Chamber of theEye

ZRep₄CT fibers/films/foams are allowed to incubate with IgG against CD45or CD34. Leucocytes are captured to ZRep₄CT scaffolds with IgG againstCD45 and mast cells are captured on ZRep₄CT scaffolds with IgG againstCD34. The cells are allowed to immobilize onto the scaffold. The cellsand the fusion protein scaffolds are transplanted into the anteriorchamber of the eye of a naked mice. The cells are inspected in vivothrough the eye window.

(B) In Vitro Studies of Non-Adherent Cells

Mast cells and leucocytes are grown on ZRep₄CT scaffolds with IgGagainst CD34 and CD45 respectively, and then monitored in vitro. Somecells are non adherent in a phase during development, e.g. neural stemcells that can grow in clusters. In the neural clusters the cells stayin a non-differentiated form. Neural stem cells are grown in clusters onZRep₄CT scaffolds with IgG against a cell receptor on the neural stemcells.

(C) Selection of Specific Cells

A ZRep₄CT scaffold (film/foam/fiber) is used for selection of cellsthrough specific antibodies. Mast cells are selected in a two stepprocedure. Step 1: Binding of cells onto a ZRep₄CT scaffolds with IgGagainst CD34. Step 2: Selection of mast cells on ZRep₄CT scaffolds withIgG against the C-kit receptor.

(D) Protein Production in Eukaryotic Cells Grown on ZRep₄CT

Many cells used for protein production are derived from non-adherentcell lines. However, large scale production is in many ways facilitatedwhen adherent cells are used, as the physical separation is facilitated.By the use of a ZRep₄CT scaffold with IgG against a cell receptor,growth of non-adherent cells can be done in adherent way.

Example 15 Investigation of the Influence of Urea Treatment on IgGBinding to Z-Rep₄CT

The effect of urea treatment of Z-Rep₄CT films and fibers on IgG bindingwas here evaluated. Films and fibers of Z-Rep₄CT were treated with ureaprior to binding of IgG from rabbit serum. Bound IgG was eluted andanalyzed by SDS-PAGE.

Fibers and films of Z-Rep₄CT, which in Example 6 were observed to bindIgG from rabbit serum and were treated with 8 M urea (200 μl 8 M urea,20 min, room temperature), were incubated with 500 μl rabbit serum (1:5dilution) for 1 h at room temperature. Films and fibers of Rep₄CT (SEQID NO: 20) were used as control material, and were treated in the sameway. After washing three times with 600 μl PBS, bound IgG was eluted in500 μl by lowering the pH to approximately 2.7 with elution buffer (0.5M acetic acid, 1 M urea, 100 mM NaCl), after which the eluted fractionswere analyzed by non-reducing SDS-PAGE (not shown).

It was concluded from the gel that films and fibers of Z-Rep₄CT retaintheir binding capacity for IgG after treatment with 8 M urea. Controlfilms and fibers of Rep₄CT did not show any IgG binding.

Example 16 Investigation of the Influence of NaOH Treatment on IgGBinding to Z-Rep₄CT

To further evaluate durability to cleaning conditions, the effect ofNaOH treatment of Z-Rep₄CT (SEQ ID NO: 14) films and fibers on IgGbinding was evaluated. Fibers and films of Z-Rep₄CT from Example 4 and5, which in Example 6 were observed to bind IgG from rabbit serum andwere treated with 8 M urea (200 μl 8 M urea, 20 min, room temperature),were now further treated with 1 M NaOH (500 μl 1 M NaOH, 20 min, roomtemperature). Films and fibers of Rep₄CT (SEQ ID NO: 20) were used ascontrol material, and were treated in the same way. After NaOHtreatment, the films and fibers were incubated with 500 μl rabbit serum(1:5 dilution) for 1 h at room temperature. After washing three timeswith 600 μl PBS, bound IgG was eluted in 500 μl by lowering the pH toapproximately 2.7 with elution buffer (i.e. 0.5 M acetic acid, 1 M urea,100 mM NaCl), after which the eluted fractions were analyzed bynon-reducing SDS-PAGE (not shown).

It was concluded from the gel that films and fibers of Z-Rep₄CT retaintheir binding capacity for IgG after treatment with 1 M NaOH. Controlfilms and fibers of Rep₄CT did not show any IgG binding.

Example 17 Quantification of IgG-HRP Binding to Z-Rep₄CT Films

(A) IgG-HRP Binding to Z-Rep₄CT Films

In order to quantify the IgG binding to Z-Rep₄CT, horseradish peroxidase(HRP) conjugated IgG (IgG-HRP) was bound to Z-Rep₄CT films.

Films of Z-Rep₄CT (SEQ ID NO: 14) and Rep₄CT (control, SEQ ID NO: 20) atdifferent concentrations (0.011-890 pmoles) were casted in 96-wellplates). The films were blocked with 100 μl 1% BSA for 1 h at roomtemperature. The films were then incubated for 1 h at room temperaturein 50 μl IgG-HRP (i.e. 34 pmole IgG-HRP, rabbit source IgG), and thefilms were washed twice with 100 μl 0.05% Tween, followed by a finalwash with 100 μl of PBS. To measure bound IgG-HRP to films, 50 μl of 50μM Amplex Red/2 mM H₂O₂ was added to one film at a time, followed bymonitoring of the absorbance at 570 nm for three minutes at a TecanPlate Reader. A dilution series of soluble IgG-HRP (0.05-0.5 pmole) wasalso measured in the same type of plate as for the films, by blockingthe wells with 100 μl 1% bovine serum albumin (BSA) for 1 h, prior toaddition of 20 μl soluble IgG-HRP, 20 μl 125 μM Amplex Red and 10 μl9.79 mM H₂O₂. Triplicate measurements were performed for the films andthe dilution series.

A linear regression fit was made using Tecan software for eachindividual measurement, corresponding to the linear region of theabsorbance at 570 nm (Abs570/min) versus time raw data plot. The slopecorresponds to the HRP conversion rate of colorless substrate to coloredproduct, and is proportional to the number of bound IgG-HRP molecules.

For each individual triplicate, a mean value and a standard deviation ofthe amount of bound IgG-HRP (pmole) was calculated. FIG. 24 A shows theamount of IgG-HRP bound to Z-Rep₄CT and Rep₄CT films of differentprotein concentrations, and FIG. 24 B shows the fractions of Z-Rep₄CTand Rep₄CT molecules in films of different protein concentrations thathave bound IgG-HRP.

For films containing 1.1 pmole of protein and more, Z-Rep₄CT films bindsignificantly more IgG-HRP than the corresponding Rep₄CT control films.The fraction of Z-Rep₄CT molecules in these films that have boundIgG-HRP is approximately ˜7% or less.

(B) IgG-HRP Binding to Z-Rep₄CT Films after NaOH Treatment

To investigate the effect of NaOH treatment on IgG binding to Z-Rep₄CTfilms, IgG-HRP was bound to NaOH treated films and the amount of boundIgG-HRP detected.

Films of Z-Rep₄CT (SEQ ID NO: 14; 1.1-890 pmole) and Rep₄CT (SEQ ID NO:20, 108 pmole) with bound IgG-HRP from Example 17 (A) were incubated in100 μl of elution buffer (pH 2.7) for 1 h at room temperature in orderto remove the bound IgG-HRP. Next, the films were incubated in 100 μl of1 M NaOH for 20-30 min at room temperature, followed by washing twice in150 μl PBS. The wells containing the films were then blocked with 1%BSA, incubated with IgG-HRP (34 pmole) and washed three times, prior toaddition of 50 μM Amplex Red/2 mM H₂O₂ and subsequent monitoring of theabsorbance at 570 nm as set out above in (A). For each individualtriplicate, a mean value and a standard deviation of the amount of boundIgG-HRP (pmole) was calculated.

FIG. 25 A shows the amount of IgG-HRP bound to Z-Rep₄CT and Rep₄CT filmsof different protein concentrations, and FIG. 25 B shows the fractionsof Z-Rep₄CT and Rep₄CT molecules in films of different proteinconcentrations that have bound IgG-HRP. FIG. 26 visualizes the amountsof bound IgG-HRP, before and after NaOH treatment, to Z-Rep₄CT andRep₄CT films.

The 108 pmole Z-Rep₄CT films show significantly more binding than thecorresponding Rep₄CT films, and the amount of Z-Rep₄CT binding ofIgG-HRP to NaOH treated films show a trend to increase as the amount ofprotein in the film increases. It seems that the amount of bound IgG-HRPto Z-Rep₄CT films is reduced approximately 2-4-fold by the harsh 1 MNaOH treatment compared to untreated films.

Example 18 Quantification of IgG-Fluorophore Binding to Z-Rep₄CT Films

Binding of IgG conjugated to a fluorophore was performed to Z-Rep₄CT andRep₄CT films containing different amounts of protein.

Films of Z-Rep₄CT (SEQ ID NO: 14) and Rep₄CT (control, SEQ ID NO: 20) atdifferent concentrations (0.011-890 pmoles) were prepared and blocked asset out in Example 17. The films were then incubated for 1 h at roomtemperature in 50 μl IgG-fluorophore (100 pmole IgG-fluorophore, rabbitsource IgG, fluorophore: Alexa Fluor 633), after which the films werewashed two times with 100 μl 0.05% Tween, followed by a final wash with100 μl of PBS. Before fluorescence measurements, 100 μl PBS was added toeach film.

A dilution series of soluble IgG-fluorophore (0-1 pmole) was alsomeasured in the same type of plate as for the films, by blocking thewells with 100 μl 1% bovine serum albumin (BSA) for 1 h, prior toaddition of 100 μl soluble IgG-fluorophore. The fluorescence wasmeasured as triplicates for films and the dilution series on a TecanPlate Reader instrument (excitation: 632 nm, emission: 660 nm, Gain:200).

For each individual triplicate, a mean value and a standard deviation ofthe amount of bound IgG-fluorophore (pmole) were calculated (FIG. 27 A)for films with 0.011-55 pmole protein. The fraction of Z-Rep₄CT andRep₄CT molecules binding IgG-fluorophore was also calculated (FIG. 27B).

It can be concluded that there is significantly more binding ofIgG-Alexa Fluor 633 to Z-Rep₄CT films than to the corresponding Rep₄CTcontrol films. No significant difference in IgG-fluorophore bindingbetween 0.011-11 pmole Z-Rep₄CT films is observed, and this does notseem to be due to autofluorescence of Z-Rep₄CT films alone at thiswavelength (data not shown). The IgG-fluorophore binding to Z-Rep₄CTfilms containing more than 55 pmole of protein could not be calculatedin any reliable way, because the fluorescence signals from those wereoutside the calibration curve.

By comparing the amounts of bound IgG-fluorophore by Z-Rep₄CT films withthe corresponding binding of IgG-HRP in Example 17, Z-Rep₄CT seems tobind more IgG-fluorophore than IgG-HRP (e.g. ˜4- and ˜6-fold differencefor 55 and 1.1 pmole films, respectively), which may be due to thedifference in size between the fluorophore and HRP. Furthermore, thefraction of Z-Rep₄CT molecules binding IgG-fluorophore also seems tohave increased compared to those of IgG-HRP binding.

Example 19 Binding of IgG from Human Blood Plasma to Z-Rep₄CT Films

In this experiment, three films of Z-Rep₄CT (SEQ ID NO: 14) wereprepared as set out in Example 3. All films had been stored, after theywere casted, for eight months in +4° C. without being immersed in anyliquid during storage. Each Z-Rep₄CT film was incubated with 500 μl ofhuman blood plasma (1:5 dilution) for 1 h at room temperature. Afterwashing three times with 600 μl PBS, bound IgG was eluted in 500 μl bylowering the pH to approximately 2.7 with elution buffer (0.5 M aceticacid, 1 M urea, 100 mM NaCl), after which the eluted fractions wereanalyzed by non-reducing SDS-PAGE (not shown). Films of Rep₄CT (SEQ IDNO: 20) were used as control material, and were treated in the same way.

It is evident from the gel that IgG (˜146 kDa) appears in the elutedfractions from Z-Rep₄CT films, indicating that films of Z-Rep₄CT haveretained the ability to bind IgG from human blood plasma after eightmonths of storage in +4° C., without being immersed in any liquid. Thecontrol films of Rep₄CT do not show any IgG in the eluted fractions.These findings extend the observations of experimental reproducibilityreported in Example 4 when using the structures according to theinvention, and also show that the protein structures can bind human IgG.

Example 20 Binding of IgG from Human Blood Plasma to an AutoclavedZ-Rep₄CT Fiber

The ability of Z-Rep₄CT to bind IgG after sterilization by autoclavetreatment was investigated. After autoclave treatment of a Z-Rep₄CTfiber, the fiber was allowed to bind IgG from human blood plasma. TheIgG binding of the autoclaved fiber was compared to that of anon-autoclaved fiber.

Two approximately equally sized Z-Rep₄CT (SEQ ID NO: 14) fibers weretransferred to two tubes containing 20 mM Tris (pH 8). One of the fiberswas then autoclaved for 20 min at 121° C. Two Rep₄CT (SEQ ID NO: 20)fibers were used as control material, and were treated in the same way.

The fibers were incubated with 500 μl of human blood plasma (1:5dilution) for 1 h at room temperature. After washing three times with600 μl PBS, bound IgG was eluted in 500 μl by lowering the pH toapproximately 2.7 with elution buffer (0.5 M acetic acid, 1 M urea, 100mM NaCl), after which the eluted fractions were analyzed by non-reducingSDS-PAGE (FIG. 28).

The gel shown in FIG. 28 was loaded according to:

(1) Human blood plasma (1:5), 1.4 μl loaded

(2) Z-Rep₄CT, non-autoclaved fiber, 14 μl loaded

(3) Z-Rep₄CT, autoclaved fiber, 14 μl loaded

(4) Rep₄CT, non-autoclaved fiber, 14 μl loaded

(5) Rep₄CT, autoclaved fiber, 14 μl loaded

(6) Molecular weight marker.

Both the non-autoclaved and the autoclaved Z-Rep₄CT fiber show a clearIgG band around 146 kDa. There is no obvious difference in the strengthof these IgG bands, suggesting that autoclave treatment has littleeffect on IgG binding ability. Neither non-autoclaved nor autoclavedfibers of Rep₄CT show any IgG in the eluted fractions. All fibers showan additional, much weaker, albumin band (˜50-60 kDa).

Example 21 Protease 3C Cleavage of Z-Rep₄CT Fibers

The Z-Rep₄CT protein (full protein architecture beingHis₆-Z-LEALFQGP-Rep₄CT; SEQ ID NO: 14) contains a Protease 3Crecognition site (LEALFQGP, Protease 3C cleaving between the amino acidsQ and G) between the Z domain and the Rep₄CT segment.

An arbitrary sized Z-Rep₄CT fiber was transferred to an Eppendorf tube,and protease cleavage was started by adding 9.6 μg of Protease 3C and0.35 μl of 1 M DTT (dithiothreitol) to a total volume of 350 μl. Thecleavage was allowed to proceed for 24 h at +4° C., after which a samplefor SDS-PAGE was withdrawn from the cleavage supernatant. The cleavagewas then allowed to proceed for another 24 h (+4° C.) when a secondsample for SDS-PAGE was withdrawn from the cleavage supernatant. The twowithdrawn samples were then analysed by SDS-PAGE (FIG. 29).

The gel shown in FIG. 29 was loaded according to:

(1), molecular weight marker

(2), supernatant of the Z-Rep₄CT fiber cleaved with Protease 3C for 24 h

(3), supernatant of the Z-Rep₄CT fiber cleaved with Protease 3C for 48h.

It can be seen from FIG. 29 that both supernatants after cleavage of aZ-Rep₄CT fiber with Protease 3C contained two distinct bands. The firstband, slightly below 35 kDa, corresponds to Protease 3C (˜31 kDa), whilethe second band is situated just above 10 kDa. Since cleavage ofZ-Rep₄CT by Protease 3C would generate two oligopeptide segmentscorresponding to (i) His₆-Z-LEALFQ (˜9 kDa) and (ii) GP-Rep₄CT (˜23kDa), it is concluded that the second band corresponds to thecleaved-off HZ fragment (9 kDa). It is therefore concluded that theProtease 3C cleavage site is available for cleavage in Z-Rep₄CT fibersand therefore, the HZ part can be removed.

Example 22 Fiber Formation of Soluble Z-Rep₄CT in the Presence of IgG

When mixing soluble Z-Rep₄CT with IgG, the kinetics of IgG binding tothe Z domains should be faster than the formation of Z-Rep₄CT fibers.The possibility to form fibers even if most of the Z domains areoccupied with IgG was studied.

Fiber Formation in the Presence of IgG

Purification of Z-Rep₄CT (SEQ ID NO: 14) was carried out in the same wayas stated earlier, and the purified protein solution was concentrated to2.2 mg/ml. Fiber formation was carried out at four different conditions,all in a total fiber forming volume of 3 ml containing 71 nmole ofsoluble Z-Rep₄CT protein. The first condition involved only Z-Rep₄CT;the second condition was Z-Rep₄CT mixed with purified rabbit IgG (8times excess of Z-Rep₄CT compared to IgG); the third condition wasZ-Rep₄CT mixed with rabbit serum (˜1.5 times excess of serum IgGcompared to Z-Rep₄CT); and the fourth condition was Z-Rep₄CT mixed withrabbit serum (˜7 times excess of Z-Rep₄CT compared to serum IgG). Fiberformation was allowed to proceed for three days in room temperature.

After three days of fiber formation, fibers had formed for conditions 1,2 and 4. In addition to a formed fiber for condition 4, a considerableamount of Z-Rep₄CT protein aggregates had also formed. For condition 3,no fiber or aggregates were visible at all.

One conclusion from this might be that fiber formation is impaired ifthe presence of lots of other biomolecules, as can be the case incondition 3, shield individual Rep₄CT molecules from interacting witheach other. Another aspect of this can be that if too much of IgG ispresent, as can be the case in condition 3, many of the Z domains inZ-Rep₄CT may have bound IgG, and a large fraction of Z-Rep₄CT with boundIgG may prevent fiber formation.

Removal of Bound IgG from Z-Rep₄CT Fibers

Fibers made at conditions 1, 2 and 4, together with the aggregates fromcondition 4 were recovered and washed in 20 mM Tris (pH 8). Next, allfibers and the aggregates were divided into two equal halves, one halffor elution of bound IgG by lowering the pH and the other half forcleavage with Protease 3C.

A first group of fibers and aggregates were transferred to Eppendorftubes and 144 μl of elution buffer (0.5 M acetic acid, 1 M urea, 100 mMNaCl), pH 2.7, was added to each tube. Elution of IgG was allowed toproceed for 30 min at room temperature, after which the elutionsupernatants were recovered and analyzed by SDS-PAGE (FIG. 30).

To a second group of fibers and aggregates, 144 μl of Protease 3C (i.e.110 μg Protease 3C), containing DTT, was added. The Protease 3C cleavagewas allowed to proceed over night at +4° C., after which the cleavagesupernatants were recovered and analyzed by SDS-PAGE (FIG. 30).

FIG. 30 displays a non-reducing SDS-PAGE gel of IgG removed fromZ-Rep₄CT fibers and aggregates formed by mixing soluble Z-Rep₄CT withIgG. The gel was loaded according to:

(1) Purified, soluble Z-Rep₄CT (2.2 mg/ml)

(2) Low pH elution of a Z-Rep₄CT fiber (condition 1)

(3) Low pH elution of a Z-Rep₄CT fiber (condition 2)

(4) Low pH elution of a Z-Rep₄CT fiber (condition 4)

(5) Low pH elution of Z-Rep₄CT aggregates (condition 4)

(6) Molecular weight marker

(7) Protease 3C cleavage of a Z-Rep₄CT fiber (condition 1)

(8) Protease 3C cleavage of a Z-Rep₄CT fiber (condition 2)

(9) Protease 3C cleavage of a Z-Rep₄CT fiber (condition 4)

(10) Protease 3C cleavage of Z-Rep₄CT aggregates (condition 4).

[Note: The molecular weight of His₆Z, Protease 3C and rabbit IgG is 9,30 and ˜146 kDa, respectively.]

It is evident form FIG. 30 that IgG is recovered from all tested fibersand aggregates regardless of under what conditions they were formed. Seealso Example 13.

Example 23 Capture of Lymphocytes to Z-Rep₄CT Using Antibody Binding

Using Z-Rep₄CT matrices, e.g. fibers and films, to bind the Fc part ofIgG, opens for the possibility for further binding of something that thecaptured IgGs are specifically directed to. One appealing thought wouldbe to isolate a certain cell type from a biological sample containingmany different cell types, using the captured IgG on the Z-Rep₄CT matrixas a cell affinity ligand. To test this cell capture approach, Z-Rep₄CTfibers and films were allowed to bind IgGs that are specificallydirected to the CD3 molecule on the cell surface of human T lymphocytes.The captured cells were analyzed by fluorescence microscopy,

Protein expression and purification of Z-Rep₄CT (SEQ ID NO: 14) werecarried out as described earlier. The purified protein was concentratedto ˜1 mg/ml, after which fibers and films were made according topreviously stated procedures (films were made in 24-well tissue cultureplates). In addition, Rep₄CT (SEQ ID NO: 20) control fibers and filmswere prepared in the same way from a ˜1 mg/ml protein solution.

To capture IgG directed towards the CD3 molecule of human lymphocytesonto the matrices, the fibers and films were immersed in 150 μl of a1:20 dilution of fluorophore conjugated anti-human CD3 IgG (mousemonoclonal IgG_(2a) to the human CD3 antigen, labeling: Alexa Fluor 488)for 1 h at room temperature. The fibers and films were washed threetimes with 300 μl of PBS (pH 7.4), after which they were analyzed forIgG binding with an inverted Nikon Eclipse Ti fluorescence microscope(excitation at 455-490 nm, detection at 500-540 nm). Both fiber and filmof Z-Rep₄CT bound the Alexa Fluor 488 conjugated IgG antibody, thusconfirming the ability of the Z domain in Z-Rep₄CT to bind IgG. Z-Rep₄CTmatrices not exposed to IgG do not show any fluorescence signal in thisselected region, and control fibers and films of Rep₄CT do not show anyfluorescence even if exposed to IgG.

Mononuclear cells (i.e. lymphocytes and monocytes) were separated fromfreshly collected human peripheral blood by gradient centrifugation atroom temperature (30 min, 400×g) in Ficoll-Paque density gradientseparation medium. The mononuclear cell fraction was recovered aftercentrifugation followed by two washes in PBS, whereafter the cells wereresuspended in 20 ml of RPMI/10% FCS medium. Monocyte depletion wasachieved by transferring the cell suspension to a T-75 tissue cultureflask, followed by incubation 90 min at 37° C. After monocyte depletion,cells in suspension were recovered and the total number of lymphocyteswas counted to 11×10⁶.

In order to bind lymphocytes to Z-Rep₄CT (SEQ ID NO: 14) matrices,lymphocytes (1 ml, ˜0.37×10⁶ cells/ml) were applied to fibers and films,followed by incubation for 30 min at +4° C. (with gentle wobbling).Next, fibers and films were washed three times with 3 ml of PBS/2% FCS(pH 7.4). Bound cells were fixated in 2% PFA (ParaFormAldehyde) for 15min at +4° C. Cell nuclei were stained by immersing fibers and filmswith bound cells in 200 μl of DAPI (1 μg/ml) staining solution for 5 minat room temperature prior to washing three times with 300 μl PBS. PBS ata volume of 300 μl was added to each fiber and film before fluorescencemicroscopy analysis using an inverted Nikon Eclipse Ti instrument(excitation at 380-395 nm, detection at 415-475 nm).

The pictures of Z-Rep₄CT fibers (not shown) show a few bound lymphocytecells for the fiber that has not been exposed to anti-human CD3 IgGantibodies, whereas the fiber that has been exposed to IgG seem to havebound more lymphocytes. In the case of Z-Rep₄CT films, stained cells areclearly visible for the film exposed to IgG, but also for the film notexposed to IgG. However, also for the films, it seems like the filmexposed to anti-human CD3 IgG prior to cell binding has more cells boundthan the corresponding film not exposed to IgG before cell binding.Moreover, control fibers and films of Rep₄CT (SEQ ID NO: 20) do not showany lymphocyte binding at all.

In this experiment, it has been shown by fluorescence microscopy thatfibers and films of Z-Rep₄CT, in contrast to those of Rep₄CT, have theability to bind a fluorescently labeled IgG antibody, namely mouseanti-human CD3 IgG. Furthermore, both Z-Rep₄CT fibers and films have theability to bind lymphocytes, regardless of possessing the IgG antibodyspecifically recognizing human T lymphocytes or not. This could implythat the Z domain itself has some affinity for human lymphocytes.However, the number of bound cells to Z-Rep₄CT matrices seems to beslightly increased when coated with IgG prior to cell binding. To beable to know if the bound cells are lymphocytes of T type, it isnecessary to apply a second antibody also directed to the human CD3molecule, in order to distinguish T lymphocytes from other types oflymphocytes (e.g. B lymphocytes and NK cells).

Example 24 Binding of Albumin from Human Plasma to Abd-Rep₄CT Films

To evaluate the accessibility of the Abd domain (residues 13-58 in SEQID NO: 16) in the films, and the ability of Abd-Rep₄CT films to bindalbumin, human blood plasma was used as albumin source. Bound albuminwas eluted and analyzed by SDS-PAGE.

Six films of Abd-Rep₄CT (SEQ ID NO: 16) prepared in Example 7 wereincubated with 500 μl of human blood plasma (1:5 dilution) for 1 h atroom temperature. After washing three times with 600 μl PBS, boundalbumin was eluted in 500 μl by lowering the pH to approximately 2.7with elution buffer (0.5 M acetic acid, 1 M urea, 100 mM NaCl), afterwhich the eluted fractions were analyzed by non-reducing SDS-PAGE (FIG.31). Films of Rep₄CT (SEQ ID NO: 20), also prepared in Example 7, wereused as control material, and were treated in the same way.

The gel shown in FIG. 31 was loaded according to:

(1) Human blood plasma (1:50), 0.7 μl loaded

(2-7) Hexaplicate of Abd-Rep₄CT, film, 14 μl loaded

(8) Empty well incubated with human blood plasma, 14 μl loaded

(9) Molecular weight marker

(10-12) Triplicates of Rep₄CT, film, 14 μl loaded.

All six films of Abd-Rep₄CT have bound albumin from human blood plasma(lanes 2-7). As only a single albumin band (˜60 kDa) appears in theeluted fraction of these Abd-Rep₄CT films, they seem to not bindanything unspecifically from the human blood plasma. Films of Rep₄CT donot show any albumin in the eluted fractions (lanes 10-12).

To investigate the stability of the films, the albumin binding abilityof Abd-Rep₄CT films that had been used once before (see above), and hadbeen stored 29 days in PBS (+4° C.) was tested again. The six films ofAbd-Rep₄CT were incubated with 500 μl of human blood plasma (1:5dilution) for 1 h at room temperature. After washing three times with600 μl PBS, bound albumin was eluted in 500 μl by lowering the pH toapproximately 2.7 with elution buffer (0.5 M acetic acid, 1 M urea, 100mM NaCl), after which the eluted fractions were analyzed by non-reducingSDS-PAGE (not shown). Films of Rep₄CT were used as control material, andwere treated in the same way.

All six films of Abd-Rep₄CT retained the ability to bind albumin fromhuman blood plasma after storage for 29 days in PBS. Films of Rep₄CT didnot show any albumin in the eluted fractions.

Example 25 Cleaning of Abd-Rep₄CT Films

(A) Urea Treatment of Abd-Rep₄CT Films

Films of Abd-Rep₄CT (SEQ ID NO: 16) (six films in total, previously usedin Example 24) were incubated with 500 μl of 8 M urea for 20 min in roomtemperature, after which they were washed three times in 600 μl PBS.Next, the films were incubated in 500 μl of human blood plasma (1:5dilution) for 1 h at room temperature. After washing three times with600 μl PBS, bound albumin was eluted in 500 μl by lowering the pH toapproximately 2.7 with elution buffer (i.e. 0.5 M acetic acid, 1 M urea,100 mM NaCl), after which the eluted fractions were analyzed bynon-reducing SDS-PAGE (not shown). All six films of Abd-Rep₄CT can stillbind albumin from human blood plasma after treatment with 8 M urea.

(B) NaOH Treatment of Abd-Rep₄CT Fibers and Films

A fiber of Abd-Rep₄CT (SEQ ID NO: 16) was first incubated in 500 μl ofhuman blood plasma (1:5 dilution) for 1 h at room temperature. Afterwashing three times with 600 μl PBS, bound albumin was eluted in 500 μlby lowering the pH to approximately 2.7 with elution buffer (0.5 Macetic acid, 1 M urea, 100 mM NaCl), after which the eluted fraction wasanalyzed by non-reducing SDS-PAGE. The same procedure was carried outfor a Rep₄CT (SEQ ID NO: 20) control fiber.

For treatment with NaOH, triplicates of three sets of Abd-Rep₄CT (SEQ IDNO: 16) films were used: (i) films previously treated with 8 M urea (see(A) above) that are treated with 1 M NaOH, followed by albumin binding;(ii) previously unused films that are treated with 1 M NaOH, followed byalbumin binding; and (iii) previously unused films that are onlyanalyzed for albumin binding. The Abd-Rep₄CT fibers used above foralbumin binding are treated with 1 M NaOH, followed by albumin bindingagain.

For treatment with NaOH, the Abd-Rep₄CT fiber and films [film sets (i)and (ii)] were incubated with 500 μl of 1 M NaOH for ˜20 min in roomtemperature, after which they were washed three times in 600 μl PBS.Next, the fiber and all three sets of films were incubated in 500 μl ofhuman blood plasma (1:5 dilution) for 1 h at room temperature. Afterwashing three times with 600 μl PBS, bound albumin was eluted in 500 μlby lowering the pH to approximately 2.7 with elution buffer (0.5 Macetic acid, 1 M urea, 100 mM NaCl), after which the eluted fractionswere analyzed by non-reducing SDS-PAGE (FIG. 32).

The gel in FIG. 32 was loaded according to:

(1) Human blood plasma (1:5), 1.4 μl loaded

(2-3, 5) Triplicate of Abd-Rep₄CT film, treated with 8 M urea and 1 MNaOH before albumin binding, 14 μl loaded

(4) Molecular weight marker

(6-8) Triplicate of Abd-Rep₄CT film, untreated before albumin binding,14 μl loaded

(9-11) Triplicate of Abd-Rep₄CT film, treated with 1 M NaOH beforealbumin binding, 14 μl loaded

(12) Abd-Rep₄CT fiber, untreated before albumin binding, 14 μl loaded

(13) Abd-Rep₄CT fiber, treated with 1 M NaOH before albumin binding, 14μl loaded

(14) Rep₄CT fiber, untreated before albumin binding, 14 μl loaded.

The Abd-Rep₄CT fiber clearly binds albumin (˜60 kDa) both before andafter treatment with 1 M NaOH (lane 12 and 13, respectively), whereasthe corresponding untreated Rep₄CT fiber does not show any albuminbinding (lane 14). All Abd-Rep₄CT films show albumin binding with noobvious difference in band strength between the untreated films and thefilms treated with 1 M NaOH before albumin binding (lanes 6-8 and 9-11,respectively). However, the films treated with both 8 M urea and 1 MNaOH before albumin binding show a decrease in the strength of theeluted albumin bands (lanes 2, 3 and 5) compared to the other two setsof films.

Example 26 Binding of Rabbit and Mouse IgG to C2-Re 4CT Films and Fibers

The accessibility of the C2 domain (residues 13-67 in SEQ ID NO: 18) inC2-Rep₄CT fibers and films was analysed as follows. Two films and onefiber of C2-Rep₄CT (SEQ ID NO: 18) were incubated with 500 μl rabbitserum (1:5 dilution), while another two films and one fiber of C2-Rep₄CT(SEQ ID NO: 18) were incubated with 500 μl of ˜50 μg/ml mouse IgG₁(monoclonal anti-rabbit immunoglobulins, mouse IgG₁ isotype, mouseascites fluid) for 1 h at room temperature. After washing three timeswith 600 μl PBS, bound IgG was eluted in 500 μl by lowering the pH toapproximately 2.7 with elution buffer (0.5 M acetic acid, 1 M urea, 100mM NaCl), after which the eluted fractions were analyzed by non-reducingSDS-PAGE (FIG. 33). Films and fibers of Rep₄CT (SEQ ID NO: 20) were usedas control material, and were treated in the same way.

The gel of FIG. 33 was loaded according to:

(1) Mouse ascites fluid (isotype IgG₁)

(2) Rabbit serum (1:50)

(3-4) Duplicates of C2-Rep₄CT, film, mouse IgG₁

(5, 7) Duplicates of C2-Rep₄CT, film, rabbit serum

(6) Molecular weight marker

(8) C2-Rep₄CT, fiber, mouse IgG₁

(9) C2-Rep₄CT, fiber, rabbit serum

(10-11) Duplicates of Rep₄CT, film, mouse IgG₁

(12) Rep₄CT, film, rabbit serum

(13) Rep₄CT, fiber, rabbit serum

(14) Rep₄CT, fiber, mouse IgG₁

[Note: Rabbit IgG is ˜146 kDa and mouse IgG is ˜160 kDa undernon-reducing SDS-PAGE conditions.]

Binding of mouse IgG₁ from ascites fluid to C2-Rep₄CT films did not giveany detectable IgG band in the eluted fractions on SDS-PAGE (lanes 3-4),but the C2-Rep₄CT fiber seems to have bound mouse IgG₁ (lane 8, mouseIgG is ˜160 kDa). However, both films (lanes 5 and 7) and the fiber(lane 9) of C2-Rep₄CT show binding of IgG from rabbit serum. As thesource of mouse IgG₁ is here in the form of an ascites fluid, it may bethe case that something in this fluid is somehow disturbing the bindingbetween C2 and IgG₁ in the film. Control films and fibers of Rep₄CT didnot show any IgG in the eluted fractions (lanes 10-14).

Example 27 Binding of IgG from Human Blood Plasma to C2-Rep₄CT Films

To further investigate the ability of C2-Rep₄CT to bind IgG, two filmsof each of C2-Rep₄CT (SEQ ID NO: 18), Z-Rep₄CT (SEQ ID NO: 14) andRep₄CT (SEQ ID NO: 20) were incubated with 500 μl of human blood plasma(1:5 dilution) for 1 h at room temperature. After washing three timeswith 600 μl PBS, bound IgG was eluted in 500 μl by lowering the pH toapproximately 2.7 with elution buffer (0.5 M acetic acid, 1 M urea, 100mM NaCl), after which the eluted fractions were analyzed by non-reducingSDS-PAGE (FIG. 34).

The gel in FIG. 34 was loaded according to:

(1) Human blood plasma (1:5), 1.4 μl loaded

(2) Molecular weight marker

(3-4) Duplicates of C2-Rep₄CT, film, 14 μl loaded

(5-6) Duplicates of Z-Rep₄CT, film, 14 μl loaded

(7-8) Duplicates of Rep₄CT, film, 14 μl loaded.

In FIG. 34, it can be seen that Z-Rep₄CT films have clearly bound IgGfrom human blood plasma (lanes 5-6). The C2-Rep₄CT films also showweaker IgG bands in the eluted fractions (lanes 3-4). The control filmsof Rep₄CT show no binding of IgG from human blood plasma (lanes 7-8).

Example 28 Comparison of Secondary Structure in Z-Rep₄CT and Rep₄CTFibers and Films Using ATR-FTIR

The secondary structure of the Z domain is α-helical in its activeconformation, while the secondary structure of Rep₄CT fibers ispredominantly of β-sheet type. If the Z domains in Z-Rep₄CT fibers andfilms are correctly folded, one would expect a higher α-helical contentin those matrices compared to in Rep₄CT fibers and films. In order toinvestigate the difference in secondary structure, fibers and films ofZ-Rep₄CT and Rep₄CT were analyzed by the spectroscopic method AttenuatedTotal Reflectance Fourier Transform InfraRed spectroscopy (ATR-FTIR), bywhich it is possible to distinguish α-helical (band position: 1648-1657cm⁻¹) from β-sheet structure (band positions: 1623-1641, 1674-1695cm⁻¹).

One film of Z-Rep₄CT (SEQ ID NO: 14) and one of Rep₄CT (SEQ ID NO: 20)were made by allowing 15 μl of protein solution to air-dry in roomtemperature over night. Fibers were made for Z-Rep₄CT and Rep₄CT andthereafter air-dried for ˜30 min in room temperature under tension.ATR-FTIR was then recorded using a platinum ATR unit from Bruker. The IRspectra for both fiber and film (not shown) show that Z-Rep₄CT has ahigher α-helical content than Rep₄CT, which indicates the presence of acorrectly folded Z domain. This is in line with maintained functionalityof the Z domain in Z-Rep₄CT structures according to the invention, seee.g. Examples 2-5, 17-19 and 22-23.

The invention claimed is:
 1. A protein structure capable of selectiveinteraction with an organic target, wherein said protein structure is apolymer comprising as a repeating structural unit a recombinant fusionprotein that is capable of selective interaction with the organic targetand comprising the moieties B, REP and CT, and optionally NT, wherein: Bis a non-spidroin moiety of more than 30 amino acid residues, whichprovides the capacity of selective interaction with the organic target,wherein the B moiety is selected from the group consisting of the Zdomain of staphylococcal protein A, staphylococcal protein A and the E,D, A, B and C domains thereof; streptococcal protein G, thealbumin-binding domain thereof and the C1, C2 and C3 domains thereof;streptavidin and monomeric streptavidin (M4); GA modules from Finegoldiamagna; and variants thereof wherein the B moiety is capable of selectiveinteraction with the organic target; REP is a moiety of from 70 to 300amino acid residues and is derived from the repetitive fragment of aspider silk protein, wherein the REP moiety is selected from the groupof L(AG)_(n)L, L(AG)_(n)AL, L(GA)_(n)L, L(GA)_(n)GL, wherein n is aninteger from 2 to 10; each individual A segment is an amino acidsequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of theamino acid residues are not Ala, and the remaining amino acid residuesare Ala; each individual G segment is an amino acid sequence of from 12to 30 amino acid residues, wherein at least 40% of the amino acidresidues are Gly; and each individual L segment is a linker amino acidsequence of from 0 to 20 amino acid residues; CT is a moiety of from 70to 120 amino acid residues and is derived from the C-terminal fragmentof a spider silk protein, wherein the CT moiety has at least 50%identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7; andNT, when optionally present, is a moiety of from 100 to 160 amino acidresidues and is derived from the N-terminal fragment of a spider silkprotein; wherein the NT moiety has at least 50% identity to SEQ ID NO: 8or at least 80% identity to SEQ ID NO:
 6. 2. The protein structureaccording to claim 1, wherein the B moiety is selected from the groupconsisting of the Z domain derived from staphylococcal protein A, andvariants thereof wherein the B moiety is capable of selectiveinteraction with the organic target.
 3. The protein structure accordingto claim 1, wherein the B moiety is selected from the group consistingof staphylococcal protein A, the E, D, A, B and C domains thereof, andvariants thereof wherein the B moiety is capable of selectiveinteraction with the organic target.
 4. The protein structure accordingto claim 1, wherein the B moiety is selected from the group consistingof streptococcal protein G, the albumin-binding domain thereof, the C1,C2 and C3 domains thereof and variants thereof wherein the B moiety iscapable of selective interaction with the organic target.
 5. The proteinstructure according to claim 1, wherein the B moiety is selected fromthe group consisting of streptavidin, monomeric streptavidin (M4) andvariants thereof wherein the B moiety is capable of selectiveinteraction with the organic target.
 6. The protein structure accordingto claim 1, wherein the B moiety is selected from the group consistingof GA modules from Finegoldia magna and protein fragments having atleast 70% identity to GA modules from Finegoldia magna.
 7. The proteinstructure according to claim 1, wherein said recombinant fusion proteinis selected from the group of proteins defined by the formulas:B_(x)-REP-B_(y)-CT-B_(z) and B_(x)-CT-B_(y)-REP-B_(z), wherein x, y andz are integers from 0 to 5; and x+y+z≧1.
 8. The protein structureaccording to claim 7, wherein said recombinant fusion protein isselected from the group of proteins defined by the formulasB_(y)-REP-CT, B_(y)-CT-REP, REP-CT-B_(E) and CT-REP-B_(E); wherein x andz are integers from 1 to
 5. 9. The protein structure according to claim8, wherein said recombinant fusion protein is selected from the group ofproteins defined by the formulas B-REP-CT, B-CT-REP, REP-CT-B andCT-REP-B.
 10. The protein structure according to claim 1, wherein saidprotein structure has a size of at least 0.1 μm in at least twodimensions.
 11. The protein structure according to claim 1, wherein saidprotein structure is in a physical form selected from the groupconsisting of fiber, film, foam, net, mesh, sphere and capsule.
 12. Theprotein structure according to claim 1, wherein the CT moiety has atleast 90% identity to SEQ ID NO:7.
 13. The protein structure accordingto claim 12, wherein the CT moiety is SEQ ID NO:7.
 14. A method forproviding a protein structure capable of selective interaction with anorganic target, wherein said protein structure is a polymer comprisingas a repeating structural unit a recombinant fusion protein that iscapable of selective interaction with the organic target and comprisingmoieties B, REP CT, and optionally NT, wherein the polymer comprisesmore than 100 fusion protein structural units, and wherein: B is anon-spidroin moiety of more than 30 amino acid residues, which providesthe capacity of selective interaction with the organic target, whereinthe B moiety is selected from the group consisting of the Z domain ofstaphylococcal protein A, staphylococcal protein A and the E, D, A, Band C domains thereof; streptococcal protein G, the albumin-bindingdomain thereof and the C1, C2 and C3 domains thereof; streptavidin andmonomeric streptavidin (M4); GA modules from Finegoldia magna; andvariants thereof wherein the B moiety is capable of selectiveinteraction with the organic target; REP is a moiety of from 70 to 300amino acid residues and is derived from the repetitive fragment of aspider silk protein, wherein the REP moiety is selected from the groupof L(AG)_(n)L, L(AG)_(n)AL, L(GA)_(n)L, L(GA)_(n)GL, wherein n is aninteger from 2 to 10; each individual A segment is an amino acidsequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of theamino acid residues are not Ala, and the remaining amino acid residuesare Ala; each individual G segment is an amino acid sequence of from 12to 30 amino acid residues, wherein at least 40% of the amino acidresidues are Gly; and each individual L segment is a linker amino acidsequence of from 0 to 20 amino acid residues; CT is a moiety of from 70to 120 amino acid residues and is derived from the C-terminal fragmentof a spider silk protein, wherein the CT moiety has at least 50%identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7; andNT, when optionally present, is a moiety of from 100 to 160 amino acidresidues and is derived from the N-terminal fragment of a spider silkprotein; wherein the NT moiety has at least 50% identity to SEQ ID NO: 8or at least 80% identity to SEQ ID NO: 6, said method comprising thesteps of: (a) providing said recombinant fusion protein; and (b)subjecting the fusion protein to conditions to achieve formation of apolymer comprising the recombinant fusion protein.
 15. An affinitymedium for immobilization of an organic target, said affinity mediumcomprising a fusion protein that is capable of selective interactionwith the organic target and comprising the moieties B, REP, CT, andoptionally NT, wherein: B is a non-spidroin moiety of more than 30 aminoacid residues, which provides the capacity of selective interaction withthe organic target, wherein the B moiety is selected from the groupconsisting of the Z domain of staphylococcal protein A, staphylococcalprotein A and the E, D, A, B and C domains thereof; streptococcalprotein G, the albumin-binding domain thereof and the C1, C2 and C3domains thereof; streptavidin and monomeric streptavidin (M4); GAmodules from Finegoldia magna; and variants thereof wherein the B moietyis capable of selective interaction with the organic target; REP is amoiety of from 70 to 300 amino acid residues and is derived from therepetitive fragment of a spider silk protein, wherein the REP moiety isselected from the group of L(AG)_(n)L, L(AG)_(n)AL, L(GA)_(n)L,L(GA)_(n)GL, wherein n is an integer from 2 to 10; each individual Asegment is an amino acid sequence of from 8 to 18 amino acid residues,wherein from 0 to 3 of the amino acid residues are not Ala, and theremaining amino acid residues are Ala; each individual G segment is anamino acid sequence of from 12 to 30 amino acid residues, wherein atleast 40% of the amino acid residues are Gly; and each individual Lsegment is a linker amino acid sequence of from 0 to 20 amino acidresidues; CT is a moiety of from 70 to 120 amino acid residues and isderived from the C-terminal fragment of a spider silk protein, whereinthe CT moiety has at least 50% identity to SEQ ID NO: 9 or at least 80%identity to SEQ ID NO: 7; and NT, when optionally present, is a moietyof from 100 to 160 amino acid residues and is derived from theN-terminal fragment of a spider silk protein; wherein the NT moiety hasat least 50% identity to SEQ ID NO: 8 or at least 80% identity to SEQ IDNO:
 6. 16. A cell scaffold material for cultivation of cells having anorganic target that is present on the cell surface, said cell scaffoldmaterial comprising the protein structure according to claim 1, whereinsaid cell scaffold material is further comprising an intermediateorganic target, wherein the B moiety is capable of selective interactionwith and is bound to said intermediate organic target, and wherein saidintermediate organic target is capable of selective interaction with theorganic target that is present on the cell surface.
 17. A method forseparation of an organic target from a sample, comprising the steps of:providing a sample containing the organic target; providing the affinitymedium according to claim 15, wherein said affinity medium is capable ofselective interaction with the organic target; contacting said affinitymedium with said sample under suitable conditions to achieve bindingbetween the affinity medium and the organic target; and removingnon-bound sample.
 18. A method for immobilization of cells, comprisingproviding a sample comprising cells of interest; applying said sample tothe cell scaffold material according to claim 16, wherein said cellscaffold material is capable of selective interaction with an organictarget that is present on the cell surface; and allowing said cells toimmobilize to said cell scaffold material by binding between the organictarget on the cell surface and said cell scaffold material.
 19. A methodfor cultivation of cells, comprising immobilizing cells of interest to acell scaffold material according to the method of claim 18; andmaintaining said cell scaffold material having cells applied theretounder conditions suitable for cell culture.